PT1372682E - Catechins for the treatment of fibrillogenesis in alzheimer`s disease, parkinson`s disease, systemic aa amyloidosis, and other amyloid disorders - Google Patents

Abstract

A method of treating an amyloid disease, or a disease characterized by alpha-synuclein or NAC fibrillogenesis, in a mammalian subject. The method includes administering to the mammal a therapeutically effective amount of a various disclosed catechins or green tea extract. A pharmaceutical composition comprising a therapeutically effective amount of a catechin and pharmaceutically acceptable excipient. The therapeutic amount of the catechin or green tea extracts is selected for efficacy in treating amyloid, alpha-synuclein or NAC fibrillogenesis in a mammalian subject.

Description

1

DESCRIPTION OF THE PREFERRED EMBODIMENTS FOR THE TREATMENT OF FIBRILOGENESIS IN ALZHEIMER'S DISEASE, PARKINSON'S DISEASE, AMYLOSIDES AND SYSTEMS AND OTHER AMYLOID DISEASES "

TECHNICAL FIELD The invention relates to compositions for the treatment of Lewy body disease or Parkinson's disease using catechins.

BACKGROUND OF THE INVENTION Alzheimer's disease is characterized by the accumulation of a peptide of 39-43 amino acids termed beta-amyloid protein or Αβ in a fibrillar form which exists as amyloid and extracellular amyloid plaques in the walls of cerebral blood vessels . It is believed that the deposition of fibrillar γβ amyloid in Alzheimer's disease is detrimental to the patient and ultimately leads to neuronal toxicity and cell death, which are hallmarks of Alzheimer's disease. Accumulated evidence implicates amyloid as an important causative factor in the pathogenesis of Alzheimer's disease. Parkinson's disease is another human disorder characterized by the formation, deposition, accumulation and / or persistence of abnormal deposits of fibrillar proteins that demonstrate many of the characteristics of amyloid. In Parkinson's disease, an accumulation of cytoplasmic Lewy bodies consisting of alpha-synuclein / NAC filaments is believed to be important in pathogenesis and as therapeutic targets. Novel agents or compounds capable of inhibiting alpha-synuclein / NAC formation, deposition, accumulation and / or persistence, or destroying alpha-synuclein / NAC preformed fibrils (or portions thereof) are considered potential therapeutic agents for the treatment of Parkinson's disease.

A variety of other human diseases also demonstrate deposition of amyloid and usually involves systemic organs (ie, organs or tissues outside the central nervous system), in which accumulation of amyloid leads to dysfunction or failure of organs. These amyloid diseases (discussed below) leading to marked accumulation of amyloid in some different organs and tissues are known as systemic amyloidoses. In other amyloid diseases, isolated organs such as the pancreas can be affected in 90% of patients with type 2 diabetes. In this type of amyloidosis, beta cells in the pancreas Langerhans is believed to be destroyed by the accumulation of fibrillar amyloid deposits consist mainly of a protein known as islet amyloid polypeptide (IAPP). Inhibition or reduction of such accumulation of amyloid is believed to lead to novel, effective treatments for type 2 diabetes. In Alzheimer's disease, Parkinson's disease and " systemic " there is currently no effective cure or treatment, and the patient usually dies within 3 to 10 years from the onset of the disease.

Amyloid diseases include but are not limited to amyloid associated with Alzheimer's disease, Down's syndrome, hereditary brain hemorrhage with Dutch-type amyloidosis, and inclusion body myositis (Askanas et al., Ann. Neurol 43: 521-560 , 1993) (in which the specific amyloid is referred to as beta-amyloid or β-protein), amyloid associated with chronic inflammation, various forms of malignancy, and Familial Mediterranean Fever (in which the specific amyloid is referred to as amyloid AA or associated amyloidosis inflammation), amyloid associated with multiple myeloma and other B cell dyscrasias (in which the specific amyloid is referred to as amyloid AL), amyloid associated with type II diabetes (wherein the specific amyloid protein is referred to as amylin or polypeptide amyloid from the islets), amyloid associated with prion diseases, including Creuzfeldt-Jakob disease, Gerstmann-Straussler syndrome, Kuru and scrapie of anim (where specific amyloid is referred to as amyloid PrP), amyloid associated with long-term hemodialysis and carpal tunnel syndrome (in which specific amyloid is referred to as beta2-microglobulin amyloid), amyloid associated with senile cardiac amyloid and Familial Amyloid Polyneuropathy (where the specific amyloid is referred to as transthyretin or pre-albumin), and endocrine-associated amyloid, such as medullary thyroid carcinoma (in which specific amyloid is referred to as procalcitonin variants).

In addition, the alpha-synuclein protein which forms fibrils, and is positive for Congo red and Thioflavin S, is found as part of Lewy bodies in the brains of patients with Parkinson's disease, Lewy body disease (Lewy in " Handbuch der Neurologie ", M. Lewandowski, ed., Springer, Berlin, pages 920-933, 1912; Pollanen et al., J. Neuropath, Exp. Neurol, 52: 183-191, 1993, Spillantini et al., Proc. , And multiple system atrophy (Wakabayashi et al., Acta Neuropath, 96: 445-803, et al., U.S.A. Acad Sci USA, 95: 6469-6473, 1998, Arai et al., Neurosis Lett.25: 83-86, 1999) 452, 1998). The discovery and identification of novel compounds or agents as potential therapeutics to halt the formation, deposition, accumulation and / or persistence of amyloid that occur in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses are desperately sought after.

DISCLOSURE OF THE INVENTION

In a first aspect, the present invention provides the use of a catechin in the preparation of a medicament for the treatment of Parkinson's disease or Lewy body disease, wherein the catechin is selected from the group consisting of catechin, epicatechin, galhocatechin gallate , epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins.

In a second aspect, the present invention provides a catechin selected from the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins, for the treatment of Parkinson's disease or Lewy body disease. The medicament may be for oral administration, parenteral injection, intravenous injection, subcutaneous injection, intramuscular injection, topical administration, or administration by aerosol spray.

In a third aspect, the present invention provides a method of treating alpha-synuclein or 5 NAC fibrillogenesis in an in vitro environment, wherein the method comprises the step of administering in the in vitro environment a therapeutically effective amount of a catechin selected from the group consists of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins.

In a fourth aspect, the present invention provides a pharmaceutical composition for use in the treatment of Parkinson's disease or Lewy body disease comprising a therapeutically effective amount of a catechin selected from the group consisting of catechin, epicatechin, galhocatechin gallate, gallate and pharmaceutically acceptable salts of the foregoing catechins, and a pharmaceutically acceptable carrier, diluent or excipient, wherein the therapeutically effective amount of the catechin comprises a dosage in the range of about 0.1 to 500 mg / kg body weight of the subject. The therapeutically effective amount of the catechin may comprise a dosage in the range of about 1.0 to 100 mg / kg body weight of the subject.

In certain embodiments, the composition comprises a mixture of two or more of the catechins selected from the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epiglacatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins. The selected catechin is preferably in substantially pure isolated form. The identification and use of standardized green tea extract and respective derivatives and constituents such as the catechin compounds shown, for example, in Figure 1, are disclosed for the therapeutic intervention of Alzheimer's disease, systemic AA amyloidosis and other amyloidoses, and diseases Parkinson's and Lewy bodies.

Additionally, methods of isolation for the identification and purification of the potent amyloid inhibitor ingredients of the green tea extract are disclosed. It is anticipated that the use of standardized green tea leaf extract and its ingredients (i.e., 50% polyphenols) contained in different commercial preparations will benefit human patients with Alzheimer's disease and other amyloidoses, and Parkinson's and Lewy bodies diseases due to the ability of the green tea leaf extract to inhibit the formation of amyloid fibrils and the formation of alpha-synuclein fibrils of Parkinson's and Lewy bodies and to cause dissolution / destruction and breakdown of amyloid and alpha fibrils -synuclein.

We disclose the identification and make the surprising discovery that commercially available catechins, such as those in standardized green tea extracts, and the standardized green tea leaf extract itself (as present in a standardized extract for 50% polyphenols) act as an impressive inhibitor of the formation of amyloid fibrils in Alzheimer's disease, formation of systemic AA amyloid fibrils, and formation of alpha-synuclein / NAC fibrils in Parkinson's disease. In addition, 7 commercially available catechins (including, but not limited to, epicatechin, catechin, galhocatechin qalate, epicatechatechin, epicatechin and / or epicatechin qalate) and standardized green tea leaf extract have the ability to cause a dismantling / destruction of pre-formed Alzheimer's type amyloid fibrils, suggesting that this may be useful for patients in later stages of Alzheimer's disease, and those afflicted with other amyloid diseases. Standardized green tea leaf extract and catechins obtained from different commercial sources (extracts isolated from gelatin coated capsules) have been found to serve as potent inhibitors of amyloid fibrillogenesis in Alzheimer's disease and protein fibrillogenesis in Parkinson's disease.

For the purposes of this disclosure, Parkinson's disease, because fibrils develop in the brains of patients with this disease (which are positive for Congo red and Thioflavin S, and which contain predominantly a secondary structure of pleated beta sheets), is identified and discussed as a disease which also exhibits the characteristics of an amyloid-like disease, it is therefore expected that the disclosures disclosed herein related to amyloidoses refer therapeutically to Parkinson's and Lewy body diseases. Thus, it is anticipated that agents or compounds found to inhibit the formation of β-amyloid fibrils in Alzheimer's disease are also effective in inhibiting the formation of alpha-synuclein / NAC fibrils. Accordingly, these agents or compounds will also serve as therapeutics for Parkinson's and Lewy bodies, as well as having efficacy as therapeutics for Alzheimer's disease, systemic amyloidosis, type 2 diabetes, and other amyloid disorders.

Selected polyhydroxylated aromatic compounds, such as epicatechin, appear to be effective as inhibitors of systemic AA amyloidosis in vivo. Significantly, we have discovered that the catechin itself (the epicatechin epimer) is not effective as an inhibitor of systemic AA amyloidosis. We have identified several polyhydroxylated aromatic compounds which inhibit the formation and / or deposition of fibrillar AA amyloid and which are useful for the treatment of systemic AA amyloidosis that occurs in patients with chronic inflammatory disorders.

Standardized green tea leaf extract and various commercially available catechins caused marked marked dose-dependent inhibition of γβ 1-40 amyloid fibril formation, determined using a Thioflavin T fluorometry assay in a dose-dependent manner. Standardized green tea leaf extract and catechins obtained from commercial sources were also potent destroyers of preformed amyloid fibrils containing β-1-42, determined using a Thioflavin T fluorometry assay, and exerted their effects in a dose-dependent manner . Selected polyhydroxylated aromatic compounds, such as epicatechin, appear to be effective inhibitors of systemic AA amyloidosis in an experimental mouse model. Finally, standardized green tea leaf extract obtained from different commercial sources caused a breakdown of ßβ 1-42 preformed amyloid fibrils into Alzheimer's disease, and catechins obtained from different commercial sources caused an inhibition of NAC-containing fibrils in disease of Parkinson's disease.

Accordingly, there is disclosed the use of standardized green tea leaf extract and / or specific catechins (in various forms, ie, a tablet, liquid form, powder form, etc.) and the respective derivatives thereof, from different sources commercial, for the treatment of fibrinoogenesis in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis and other amyloidoses. Also disclosed are methods of isolating to identify and purify the key amyloid inhibitor ingredients in the green tea extract material. It is anticipated that the identification of the " active ingredients " inhibitors in plant extracts extracted from green tea lead to the design of new drugs for anti-amyloid therapies of the future. It is anticipated that standardized use of standardized green tea leaf extract and its ingredients, such as catechins contained in different commercial preparations, will benefit human patients at all stages of Alzheimer's, Parkinson's and other amyloid or amyloid-like diseases, due to demonstrated ability of catechins and standardized green tea extract to inhibit the formation of β-amyloid fibrils and the formation of alpha-synuclein fibrils (important for the treatment of early to intermediate stages of Alzheimer's disease and Parkinson's disease, respectively ), and cause dissolution / destruction and disintegration of preformed amyloid fibrils (important for the treatment of intermediate to late stages of Alzheimer's disease and Parkinson's disease, respectively). Similarly, catechins and standardized green tea leaf extract are anticipated to benefit patients with different systemic amyloid diseases, such as systemic AA amyloidosis, and type 2 diabetes, irrespective of the phase of accumulation of amyloid and organ (or tissue) involved. Although the results of the tea extract are exemplified with the Species Camellia sinensis, extracts of other species of the family Theaceae are believed to have similar effects. The use of green tea, green tea leaves and extracts, and catechins or their derivatives, is disclosed for the treatment of the formation, deposition, accumulation and / or persistence of amyloid and / or fibrils in Alzheimer's disease, Parkinson's disease, amyloidosis Systemic AA, type 2 diabetes and other amyloidoses.

It is also intended that " treatment ", in all possible cases, include and encompass " in vitro treatment ", either for experimental or screening purposes, and whether in vitro treatment leads or is not intended to conduct or not , treatment of a similar fibrilogenesis, or any amyloid or alpha-synuclein disease corresponding to such fibrilogenesis, in a mammalian subject.

Also disclosed is the use of green tea, leaves and green tea extracts or their derivatives, of the species Camellia sinensis for the treatment of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis , type 2 diabetes and other amyloidoses.

Green tea, leaves and extracts of green tea or its derivatives, from the family Theaceae are disclosed for the treatment of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type diabetes 2 and other amyloidoses. Capsules, tablets, caplets, soft and hard gelatin capsules, candies, cachets, cachets, vegetable capsules, liquid drops, elixirs, suspensions, emulsions, solutions, syrups, tea bags, (in the form of a solid or in a liquid medium), suppositories, sterile injectable solutions, sterilized packaged powders and / or powdered powders available on the market which contain green tea, green tea leaves and extracts or their derivatives, are disclosed for the treatment of patients with Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Green tea, leaves and extracts of green tea or its derivatives, and / or polyphenols contained in green tea, leaves and extracts of green tea or their derivatives, for the treatment of the formation, deposition, accumulation and / or persistence of amyloid in disease Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses. The invention is described with reference to specific embodiments, species and parts of plants, methods and procedures and the like. However, it will be recognized by those skilled in the art that various chemical substitutions may be made on the disclosed compounds without departing from the spirit and scope of the invention. In particular, it is known that catechins can be isolated and / or purified from plant materials by some different methods. It will further be recognized that these alternative methods, and consequent changes in other method steps, such as the use of different solvents or different columns for purification, and catechins of a partially purified polyphenol composition, fall within the scope of the presently disclosed extracts derived from plants and compounds.

Also disclosed is the use of catechins contained in green tea, green tea leaves and extracts or their derivatives, for the treatment of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Catechins, including but not limited to catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and / or epicatechin gallate, whether contained in green tea, green tea leaves or extracts thereof or derived from other sources natural or synthetic, are disclosed for the treatment of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Also disclosed is the use of bioflavanoids contained in green tea, green tea leaves and extracts or their derivatives, for the treatment of the formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses, and the use of 13 flavanols contained in green tea, green tea leaves and extracts or their derivatives, for the treatment of the formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Also disclosed is the use of flavanodiols contained in green tea, green tea leaves and extracts or their derivatives, for the treatment of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses, and the use of flavanoids contained in green tea, green tea leaves and extracts or derivatives thereof, for the treatment of the formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Also disclosed is the use of tannins contained in green tea, green tea leaves and extracts or their derivatives, for the treatment of the formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses.

Also disclosed are methods of isolating the active ingredients present in green tea, green tea leaves and extracts or their derivatives, for use as potent agents that inhibit amyloid formation, amyloid deposition, amyloid accumulation, persistence of amyloid, cause a dismantle / destroy and / or cause a dismantling of preformed or pre-deposited amyloid fibrils in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses. Methods of isolating the active ingredients of green tea, green tea leaves and extracts or their derivatives include the application of some common techniques known to practitioners, including but not limited to thin layer chromatography using silica coated plates, and separation and isolation using high or low pressure liquid chromatography (HPLC). Other active ingredients of green tea, green tea leaves and extracts or their derivatives which prove to be potent inhibitors of amyloid formation, amyloid deposition, amyloid accumulation, persistence of amyloid, and / or cause dismantling / destruction and disintegration of preformed or pre-deposited amyloid fibrils in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses, are identified by re-testing individual bands or fractions (separated by thin layer chromatography, flash chromatography column and / or HPLC) using specific assay tests described in the Examples. Sufficient insulation of these active ingredients contained in individual bands and / or fractions is then sent for specific analyzes, which may include but are not limited to scanning electron microscopes equipped with a dispersive energy x-ray analyzer to detect and spatially map some elements present in each sample, elemental combustion analysis to determine the relative% of carbon, hydrogen and nitrogen, high resolution mass spectrometry to determine molecular weight and elemental composition, infrared spectroscopy by Fourier transform to determine functional groups and make comparisons with the spectra of 15 known compounds, differential scanning calorimetry for determination of melting point, atomic absorption, gel chromatography, high performance liquid chromatography, proton nuclear magnetic resonance spectroscopy and C for characterization of the material and for prop to prescribe information regarding the position of atoms relative to each other, and UV / VIS spectroscopy. Further techniques are expected to be developed as part of the additional isolation of potent active ingredients from green tea, green tea leaves and extracts or their derivatives. The use of green tea, leaves and extracts of green tea or its derivatives, and / or its ingredients [(regardless of the commercial source and regardless of the final form for human consumption, i.e., pills, tablets, " caplets ", hard capsules, cachets " capsules, vegetable capsules, liquid droplets, elixirs, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or liquid medium), suppositories, sterile injectable solutions, sterile packaged powders and / or tea powder sheets] is disclosed for inhibition of amyloid formation, deposition, accumulation and / or persistence, irrespective of their clinical setting.

Also disclosed are compositions and methods involving the administration to a subject of a therapeutic dose of green tea, green tea leaves and extracts or their derivatives, which inhibits the deposition of amyloid. Accordingly, the disclosed compositions and methods are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The compounds disclosed herein may be used therapeutically to treat amyloidosis, or may be used prophylactically in a subject susceptible to amyloidosis. The disclosed methods rely, at least in part, on direct inhibition of the formation of amyloid fibrils, causing disruption / destruction and / or disintegration of preformed amyloid fibrils.

Also disclosed are pharmaceutical compositions for the treatment of amyloidosis. The pharmaceutical compositions disclosed include a therapeutic compound in an amount effective to inhibit the deposition of amyloid and a pharmaceutically acceptable carrier.

Also disclosed is the use of any and all synthetic compounds which are similar to green tea, green tea leaves, their extracts or derivatives and / or their active ingredients, for use as potent agents that inhibit amyloid formation, amyloid deposition , accumulation of amyloid, persistence of amyloid, cause a dismantling / destruction and / or disintegration of preformed or pre-deposited amyloid fibrils in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses. An isolation method is disclosed for purifying and identifying the amyloid inhibiting ingredients of green tea, green tea leaves and extracts or their derivatives. In one such method, an extract prepared from pills, caplets, soft and hard gelatin capsules, candies, sachets, cachets, vegetable capsules, liquid droplets, elixirs, suspensions, emulsions, solutions, syrups, tea bags, aerosols (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterilized packaged powders, commercially available tea powders, using the method employing some or all of the following steps : a) extracting from green tea, leaves and extracts of green tea or its derivatives, regardless of the way described above, using water or alcohol (i.e., methanol, ethanol or propanol, b) centrifuging at 2500 x g for 20 minutes and collection of the supernatant, c) rotary evaporation to dryness for compounds extracted with alcohol and lyophilization for compounds extracted with water, d) washing of the dry powder obtained with 4 volumes of petroleum ether (repeated 4 times), followed by centrifugation (each time) at 2500 x g for 20 minutes, and collection of supernatants and granules, e) air drying of collected granules, f) (g) lyophilization of collected supernatants (referred to as aqueous extracts), (h) redissolution of the granules or lyophilized aqueous extract powder in acetonitrile / water / trifluoroacetic acid (TFA) , i) injection and separation by HPLC or low pressure chromatography, j) identification of amyloid inhibitor ingredients by performing tests in relevant in vitro and in vivo assays, and k) sending for structural analysis and elemental composition as described on here.

Also disclosed is a composition in the form of a dietary supplement for providing, supporting or ameliorating in a subject one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities consisting of nutritional support of cognitive decline or memory related to the age, normal brain function, cognitive ability, and concentration, wherein the composition comprises green tea, leaves and extracts of green tea or its derivatives. A composition in the form of a dietary supplement is disclosed to promote, maintain or intensify in a subject one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities consisting of mental acuity, alertness, well-being cognitive function, normal brain function, cognitive ability, mental performance, memory concentration, mental acuity, mental vitality, mental clarity, short term memory, normal brain function, learning, and good brain health, wherein the composition comprises green tea, leaves and extracts of green tea or its derivatives.

Also disclosed is a composition, in the form of a dietary supplement, for promoting or supporting a healthy pancreatic function in a subject by assisting in promoting the normal function of insulin, or in reducing, destroying, dissolving, inhibiting, eliminating or preventing in a subject a or more states involving the pancreas selected from the group of states involving the pancreas consisting of deposits of amyloid fibrils, amyloid protein deposits, pancreas associated with amyloid fibril deposits, amyloid protein deposits associated with the pancreas, formation and growth of amyloid fibrils, and formation and growth of pancreatic amyloid fibrils, wherein the composition comprises green tea, green tea leaves and extracts or their derivatives. The use of (a) epicatechin or its derivatives, (b) catechins, hydrated catechin or derivatives thereof, (c) galhocatechin gallate or derivatives thereof, (d) epicatechin gallate or derivatives thereof, (e) epigalhocatechin or (g) green tea, leaves and extracts of green tea or its derivatives, (h) green tea, leaves and extracts of green tea or its derivatives, of the species Camellia sinensis, (i) green tea, leaves and extracts of green tea or its derivatives, from Theaceae family, (j) capsules, tablets, " caplets ", soft and hard gelatin capsules, candies, sachets, cachets " (as a solid or in a liquid medium), suppositories, sterile injectable solutions, sterile injected powders, sterile injectable solutions, sterile injectable solutions, sterile injectable solutions, and (k) catechins, including but not limited to catechin, epicatechin, epigalhocatechin, epicatechin gallate, and the like, and / or powdered products available on the market which contain green tea, green tea leaves and extracts, , epigalhocatechin gallate and galhocatechin gallate contained in green tea, green tea leaves and extracts or derivatives thereof, for the treatment of formation, deposition, accumulation and / or persistence of fibrils in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis , type 2 diabetes and other amyloidoses.

Also disclosed are compositions and methods involving the administration to a subject of a therapeutic dose of 20 catechins or derivatives thereof, which inhibits the deposition of amyloid. Accordingly, the disclosed compositions and methods are useful for inhibiting amyloidosis in disorders in which amyloid deposition occurs. The compounds may be used therapeutically to treat amyloidosis or may be used prophylactically in a subject susceptible to amyloidosis. The methods rely, at least in part, on the direct inhibition of the formation of amyloid fibrils, causing disruption / destruction and / or disintegration of preformed amyloid fibrils. The use of any and all synthetic catechins or their derivatives and / or their active ingredients, for use as potent agents which inhibit amyloid formation, amyloid deposition, amyloid accumulation, amyloid persistence, cause a dismantling / destruction are disclosed. and / or disintegration of preformed or pre-deposited fibrils in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses. A pharmaceutical agent for the treatment of an amyloid disease in a patient is disclosed, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the family Theraceae, and in particular of the genus Camellia. The pharmacological agent preferably comes from a plant of the genus Camellia, species sinensis. The pharmacological agent is preferably an extract obtained from Camellia sinensis, wherein the extract comes from the dried leaves. The pharmacological agent preferably has a therapeutically effective amount of standardized green tea leaf extract or specific catechins at a dosage in the range of from about 0.1 to about 500 mg / kg body weight of the patient, and more preferably in the range of from about 1.0 to about 100 mg / kg patient body weight.

Preferred pharmaceutical agents have a percent by weight of vegetable extract in the agent in the range of from about 70% to about 95%, and may also have a pharmaceutically acceptable carrier, diluent or excipient. Preferably, the pharmaceutical agent has an amyloid inhibitory activity or efficacy greater than 50%.

The disclosed compositions also have the ability to reduce, eliminate, prevent, inhibit, disrupt, disrupt or disintegrate deposits of fibrils or amyloid proteins, deposits of brain-associated amyloid fibrils or brain-associated amyloid protein deposits, as well as formation of amyloid fibrils , or formation of age-associated amyloid fibrils, formation of brain-associated amyloid fibrils; as a result, will promote mental acuity, promote mental alertness, provide nutritional support for cognitive or memory-related or age-related decline, promote cognitive well-being, support brain function, improve cognitive ability, performance mental or memory, promote concentration and mental acuity, improve mental vitality, promote greater mental clarity and alertness, improve short-term memory, reduce or reverse cognitive decline or memory associated with age, support normal brain function , intensify learning or memory; improve concentration, intensify mental performance, reduce mental decline, reduce the likelihood of occurrence of age-related brain disorders, and maintain good brain health. It is anticipated that the disclosed compositions also have the ability to reduce, eliminate, prevent, inhibit, disrupt, disrupt or disrupt deposits of fibrils or amyloid proteins, fibril deposits or amyloid proteins associated with the pancreas, as well as the formation and growth of amyloid fibrils , and formation and growth of amyloid fibrils associated with the pancreas; as a result, will support healthy pancreatic function and promote pancreatic function by helping to promote normal insulin function.

The disclosed compositions may also include carriers, diluents and / or excipients commonly used in the pharmaceutical and dietary supplements industries, and any such additions, known to those skilled in the art, are acceptable and may be employed without departing from the scope of the invention.

Also disclosed is a method of isolating amyloid-inhibiting constituents in green tea, leaves, green tea extracts or derivatives thereof, which method comprises the following steps: a) extraction of green tea, leaves, green tea extracts or derivatives thereof with water, or with an organic solvent, b) removal of insoluble materials, c) evaporation to dryness or lyophilization to obtain a powder, d) recovery and redissolution of the amyloid inhibitor constituents obtained in the water or organic solvent, and e) injection and Separation by high pressure or low pressure liquid chromatography.

Representative constituents of green tea include catechins, bioflavanoids, flavanols, flavanodiols, flavanoids, tannins or derivatives thereof, although for purposes of this disclosure these substances may also optionally be synthetically derived or independently found in other plant sources. The extraction step of the vegetable matter with an organic solvent also comprises adding methanol initially to plant materials which are in powder form, and the resulting mixture is stirred overnight. The solvent used in the extraction step of amyloid inhibiting ingredients preferably has a polarity ranging from that of water to that of pentanol. The removal step of insoluble materials is preferably effected by centrifugation of the extract and collection of the supernatant. The concentration step of the extract is preferably effected by rotary evaporation or lyophilization (for aqueous extracts). After the extraction and centrifugation steps, the extraction and centrifugation procedure is preferably repeated further 1 to 5 times, and the supernatants are collected.

Following the repeated extraction and concentration steps, the supernatants are preferably pooled and dried using a rotary evaporator or lyophilization (for aqueous extracts). The dry powder is washed with 4 volumes of petroleum ether (repeated 4 times), followed by centrifugation (each time) at 2500 x g for 20 minutes, and collection of the supernatants and granules. The collected granules are air-dried and extracted again with water and centrifuged at 2500 g for 20 minutes. The collected supernatants (referred to as aqueous extracts) are lyophilized, and the granules or lyophilized powdered aqueous extract are redissolved in acetonitrile / water / trifluoroacetic acid (TFA) for HPLC injection or low pressure chromatography. The dissolved granule is divided into equal portions and is injected into an HPLC. HPLC preferably contains a 1 x 25 cm Cis column, although other sizes may serve, and is maintained at 30 ° C with a flow rate of 2 ml / minute. The sample portions injected into the HPLC are eluted with gradients of A and B, such as 0% B for 5 minutes, 0-15% B for 5-10 minutes, 15-45% B for 10-70 minutes, and 45-100% B for 70-85 minutes; in which B = 95% acetonitrile with 0.5% acetic acid in distilled water, and A = 5% acetonitrile with 0.5% acetic acid in distilled water. HPLC eluents are monitored at all wavelengths, 4 ml fractions are collected in a collector of fractions and pooled peaks are obtained at various retention times (from 0 to 85 minutes). The fractions obtained can be concentrated by lyophilization after most of the acetonitrile is removed by rotary evaporation.

Thereafter, the concentrated fractions obtained are tested in relevant in vitro assays to identify potent inhibitors of amyloid fibril formation, or dismantling / destruction or disintegration of preformed amyloid fibrils. The amyloid inhibiting ingredients are preferably collected from the approximate HPLC retention times of 10-70 minutes.

Also disclosed is a method for the treatment of an amyloid disease in a patient comprising the step of administering to the patient a therapeutically effective amount of green tea, green tea leaves and extracts or their derivatives. Green tea, leaves and extracts of green tea or its derivatives, are preferably administered orally or by aerosol spray or in a parenterally injectable or infusible form.

In disclosed methods, the formation, deposition, accumulation and / or persistence of amyloid in a subject is inhibited by administration to the subject of a therapeutic dose of the invention. The term " subject " include living organisms in which amyloidosis may occur. Examples of subjects include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats and respective transgenic species. Administration of green tea, leaves and green tea extracts or their derivatives to a subject to be treated may be effected using known procedures, in dosages and for periods of time effective to inhibit the formation, deposition, accumulation and persistence of amyloid in the subject. An effective amount of the therapeutic compound necessary to obtain a therapeutic effect may vary according to factors such as the amount of amyloid already deposited at the clinical site in the subject, the subject's age, gender and weight, and the ability of the therapeutic compound to inhibit amyloidosis in the subject. A method of treating, preventing or managing an amyloidosis in a mammalian subject susceptible or affected by amyloidosis is disclosed which method comprises the step of administering to the subject a therapeutic amount of a plant source of a tea source green, 26 green tea leaves, standardized green tea extract, or derived from green tea. There is provided a method of treating, inhibiting, preventing or managing the formation, deposition, accumulation, aggregation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses in a mammalian subject , which method comprises the step of administering to the subject a therapeutic amount of a substance selected from the group of substances consisting of green tea, green tea leaves, standardized green tea extract, green tea derivative, catechins, bioflavanoids, flavanols, flavanodiols , flavanoids, tannins or derivatives thereof. The substance is preferably a catechin selected from the group of catechins consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, or a derivative of one of the above group.

In any of these embodiments, in the step of administering vegetable matter, a therapeutic amount of one or more plant materials selected from the group of plants consisting of and commonly known as Cat's Claw, gingko biloba, rosemary, gotu kola, bacopin and ginseng. There is provided a method of treating, inhibiting, preventing or managing the formation, deposition, accumulation, aggregation and / or persistence of alpha-synuclein 27 fibrils in Parkinson's disease, Lewy body disease, or multiple system atrophy in a which method comprises the step of administering to the subject a therapeutic amount of a substance selected from the group of substances consisting of green tea, green tea leaves, standardized green tea extract, green tea derivative, catechins, bioflavanoids, flavanols , flavanodiols, flavanoids, tannins or derivatives thereof. The substance is preferably a catechin selected from the group of catechins which consist of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin, and epicatechin gallate, or a derivative of one of the above group. There is provided a method of promoting the mental alertness in a patient, which method comprises the step of administering to the patient a therapeutically effective amount of plant matter of a plant of the family Theaceae, and preferably of a plant of the genus Camellia, species sinensis. This method can also be used to inhibit the formation of amyloid deposits in the brain. A method is presented to promote, maintain or intensify in one patient one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities consisting of mental acuity, mental alertness, cognitive well-being, normal brain function, cognitive ability , mental performance, memory, concentration, mental acuity, mental clarity, short term memory, normal brain function and learning, which method comprises the step of administering to the subject a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. There is provided a method of providing, supporting or ameliorating in a patient one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities which consist of normal brain function, cognitive ability, and concentration, which method comprises the step of administering to the patient a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. We present a method of reducing in one patient one or more of the mental or cognitive effects selected from the group of mental or cognitive effects that consist of cognitive or memory decline associated with age, mental decline, and likelihood of related brain or cognitive disorders with age, which method comprises the step of administering to the patient a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. There is provided a method of reducing, destroying, dissolving, inhibiting, eliminating or preventing in a patient one or more states involving the brain selected from the group of states involving the brain consisting of deposits of amyloid fibrils, amyloid protein deposits, amyloid fibrillar deposits associated with the brain, deposits of brain-associated amyloid proteins, formation and growth of amyloid fibrils, formation and growth of age-associated amyloid fibrils, formation and growth of brain-associated amyloid fibrils, which method comprises the step of administering to the patient an therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. A method for promoting or supporting a healthy pancreatic function in a patient is provided by assisting in promoting a normal insulin function, which method comprises the step of administering to the patient a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis . There is provided a method of reducing, destroying, dissolving, inhibiting, eliminating or preventing in a patient one or more states involving the pancreas selected from the group of states involving the pancreas consisting of deposits of amyloid fibrils, amyloid protein deposits, amyloid fibril deposits associated with the pancreas, amyloid protein deposits associated with the pancreas, formation and growth of amyloid fibrils, formation and growth of pancreatic associated amyloid fibrils, which method comprises the step of administering to the patient a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis.

The use of a source of green tea, green tea leaves or standardized green tea leaf extract or derivatives thereof in the preparation of a pharmaceutical composition or dietary supplement for the treatment, prevention and / or management of an amyloidosis in a mammalian subject susceptible or affected by amyloidosis. The use of a source of green tea, green tea leaves, standardized green tea leaf extract or derivatives thereof in the preparation of a pharmaceutical composition or dietary supplement to inhibit the formation, deposition, accumulation or persistence of fibrils amyloid or cause dissolution / destruction or disintegration of preformed amyloid fibrils. There is provided a pharmaceutical composition or dietary supplement for the treatment, prevention, or management of amyloidosis in a mammalian subject susceptible or affected by amyloidosis, the composition of which comprises a source of green tea, green tea leaves or standardized green tea leaf extract, or and, if desired, a pharmaceutically or dietary acceptable carrier, diluent or excipient. There is provided a pharmaceutical composition or dietary supplement for inhibiting the formation, deposition, accumulation or persistence of amyloid fibrils or cause dissolution / destruction and / or breakdown of preformed amyloid fibrils, the composition of which comprises a source of green tea, green tea leaves or standardized green tea leaf extract, or derivatives thereof, and, if desired, a pharmaceutically or dietary acceptable carrier, diluent or excipient. The use of catechins, bioflavanoids, flavanols, flavanodiols, flavanoids, tannins or derivatives thereof for the treatment, prevention or management of formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, AA amyloidosis systemic, type 2 diabetes and other amyloidoses, in a mammalian subject susceptible to amyloidosis. There is provided a pharmaceutical composition or dietary supplement for the treatment, prevention or management of the formation, deposition, accumulation and / or persistence of amyloid in Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses in a mammalian subject susceptible to this amyloid state, wherein the composition comprises catechins, bioflavanoids, flavanols, flavanodiols, flavanoids, tannins, or derivatives thereof, and, if desired, a pharmaceutically or dietetically acceptable carrier, diluent or excipient. There is provided a pharmaceutical composition or dietary supplement for the treatment, inhibition, prevention or management of the formation, deposition, accumulation, aggregation and / or persistence of alpha-synuclein fibrils in Parkinson's disease, Lewy body disease, or multiple atrophy systems in a mammalian subject which composition comprises a substance selected from the group of substances consisting of green tea, green tea leaves, standardized green tea extract, green tea derivative, catechins, bioflavanoids, flavanols, flavanodiols, flavanoids, tannins, or respective derivatives. The use of a source of green tea, green tea leaves or standardized green tea leaf extract or derivatives thereof in the preparation of a pharmaceutical composition or dietary supplement to provide, support or ameliorate in a subject one or more of the qualities mental or cognitive. The use of a source of green tea, green tea leaves or standardized green tea leaf extract or derivatives thereof in the preparation of a pharmaceutical composition or dietary supplement to promote or support healthy pancreatic function in a subject is presented. There is provided a pharmaceutical composition or dietary supplement for providing, supporting or ameliorating in a subject one or more of the mental or cognitive qualities comprising a source of green tea, green tea leaves or standardized green tea leaf extract, or derivatives thereof. There is provided a pharmaceutical composition or dietary supplement for promoting or supporting a healthy pancreatic function in a subject comprising a source of green tea, green tea leaves or standardized green tea leaf extract or derivatives thereof. A pharmacological agent is presented to promote, maintain or intensify in a patient one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities that consist of mental acuity, mental alertness, cognitive well-being, normal brain function, ability cognitive, mental performance, memory, concentration, mental acumen, mental vitality, mental clarity, short term memory, normal brain function, and learning, and good brain health, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. A pharmacological agent is provided to provide, support or ameliorate in a patient one or more of the mental or cognitive qualities selected from the group of mental or cognitive qualities which consist of nutritional support of cognitive decline or memory related to age, normal brain function, cognitive ability, and concentration, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. A pharmacological agent is presented to reduce in one patient one or more of the mental or cognitive effects selected from the group of mental or cognitive effects which consist of cognitive decline or memory related to age, mental decline, and likelihood of occurrence of brain or cognitive disorders related diseases, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. There is provided a pharmacological agent for reducing, destroying, inhibiting or preventing in a patient one or more states involving the brain selected from the group of states involving the brain consisting of amyloid fibrillar deposits, amyloid protein deposits, associated amyloid fibrillar deposits brain deposits, β-brain deposits, brain-associated amyloid deposits, brain amyloid deposits, formation and growth of amyloid fibrils, formation and growth of age-associated amyloid fibrils, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. A pharmacological agent for promoting or supporting a healthy pancreatic function in a patient is provided by assisting in promoting a normal insulin function, wherein the pharmacological agent comprises a therapeutically effective amount of plant matter of a plant of the genus Camellia, species sinensis. There is provided a pharmacological agent for reducing, destroying, inhibiting or eliminating or preventing in a patient one or more conditions involving the pancreas selected from the group of states involving the pancreas consisting of deposits of amyloid fibrils, deposits of amyloid proteins, deposits of fibrils amyloid deposits of islets, amyloid polypeptide deposits of the islets, deposits of amyloid proteins associated with the pancreas, formation and growth of amyloid fibrils, formation and growth of pancreatic associated amyloid fibrils, wherein the pharmacological agent comprises a therapeutically amount effective plant matter of a plant of the genus Camellia, species sinensis.

For a more detailed discussion and background information regarding amyloid and amyloidosis, Alzheimer's disease and the aging population, amyloid as a therapeutic target for Alzheimer's disease and Parkinson's disease and formation of alpha-synuclein fibrils see our application for U.S. patent application 20030017998 and registered patents 6,037,327, 6,264,994 and 6,346,280. 35

These and other features and advantages will become more apparent when the following detailed description is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a set of structures for catechin derivatives. FIGURE 2 is a black-and-white chart of a Thioflavin T fluorometry assay used to determine dose-dependent effects of standardized green tea leaf extract on the inhibition of γβ 1-40 amyloid fibril formation in Alzheimer's disease. FIGURE 3 is a black-and-white chart of a Thioflavin T fluorometry assay used to determine the dose-dependent effects of standardized green tea leaf extract in the dismantling / destruction of preformed Αβ 1-42 amyloid fibrils in Alzheimer's disease. FIGURE 4 is a black and white graph of a spectrophotometric assay of Αβ with Congo red to determine the effects of standardized green tea leaf extract (from 2 commercial sources) on the breakdown of preformed Αβ 1-42 fibrils into disease of Alzheimer's disease. FIGURE 5 is a black and white graph of a Thioflavin T fluorometry assay used to determine the dose-dependent effects of different catechins available on the market in inhibiting the formation of β-1-40 amyloid fibrils in Alzheimer's disease. FIGURE 6 is a color photomicrograph demonstrating the inhibition of β-1-40 fibrillar formation in Alzheimer's disease by epicatechin. FIGURE 7 is a black-and-white chart of a Thioflavin T fluorometry assay used to determine the dose-dependent effects of commercially available catechins in the dismantling / destruction of preformed Αβ 1-42 amyloid fibrils in Alzheimer's disease. FIGURE 8 is a black and white graph of a Thioflavin T fluorometry assay used to determine the dose-dependent effects of different catechins available on the market in inhibiting the formation of NAC fibrils in Parkinson's disease (also referred to as " NAC -P "). FIGURE 9 shows black and white photomicrographs of amyloid deposition in the spleen in an experimental AA amyloidosis mouse model. FIGURE 10 shows black and white photomicrographs of amyloid deposition in the liver in an experimental amyloidosis AA mouse model. FIGURE 11 is a graph demonstrating the effect of specific phenolic compounds on the destruction / dismantling of preformed amyloid fibrils consisting of islet amyloid polypeptide (IAPP). FIGURE 12 is a circular dichroism spectroscopy graph demonstrating the destruction of β-sheet structure in Αβ 1-42 amyloid fibrils by epicatechin. FIGURE 13 is a graph demonstrating the inhibition of epicatechin, but not hydrated catechin, splenic amyloid in an experimental model of systemic AA amyloidosis mouse. FIGURE 14 shows black and white photomicrographs of inhibition of epicatechin splenic AA amyloid deposition in a systemic AA amyloidosis mouse model. FIGURE 15 illustrates the structures of some of the polyhydroxylated aromatic-containing compounds designed to have anti-amyloid activity.

BEST MODE FOR IMPLEMENTING THE INVENTION

Turning now to the Figures and Examples, the invention will be described in preferred embodiments by reference thereto.

Some Definitions " Pharmaceutically acceptable analogs & derivatives ". Pharmaceutically acceptable analogues and derivatives of a claimed compound include various R group type substitutions, and any other structural modifications derived therefrom which do not affect the disclosed efficacy of these compounds. " Percentage of Purity ". The disclosed compositions contain one or more catechins or green tea derivatives, wherein each catechin or green tea derivative is present in the composition in a percentage or percentage purity ratio which " significantly exceeds " a percentage proportion of the natural presence of the same substance in a plant, or in a plant extract. For example, suppose a particular catechin is present in a plant in a percentage by weight of 0.01 percent, and is present in a plant extract in a percentage by weight of 1.0 percent. Then, in a disclosed composition, the same catechin is present in the composition in a percentage by weight which is significantly greater than 0.01 percent or 1.0 percent, say 10 percent. Following this line, other proportions may also be applied as a percentage of the composition or percent presence by volume, or percent purity. By way of further example, without limiting the scope of the invention to this or other examples, a catechin is present in a tablet to be delivered orally in accordance with the disclosure disclosed herein. Catechin is an isolated catechin present in a 98.5% purity percentage (i.e., catechin is 98.5% pure as measured by conventional purity indicia such as, for example, the characteristic isolated abrupt peak band on HPLC). However, the particular catechin is a tablet ingredient present only at 15%. It is known that catechin is present in a plant in a dry weight percentage of 0.06, while it is known that certain extracts of the same plant contain up to 0.75 percent dry weight of the same catechin. In this example, catechin is proportionally more present in the tablet than in the extract for a ratio of 20: 1, and this is a measure that significantly exceeds the presence in natural percentage ratio in a plant or plant extract. 39

In general, a catechin is present in a therapeutically administered dose form having a percentage of catechin (by weight, dry weight, volume, or purity) that is 10-fold (or greater) greater than the natural percentage presence of the same catechin in a plant is a percentage that " significantly exceeds " the natural percentage presence of the catechin in the plant. In this context, when speaking of extracts from a plant, it should be noted that only conventional or natural extracts (juices, concentrates and the like, or known extracts and used for other purposes), and not new extracts prepared after the date of priority of this disclosure, from which it is intended to concentrate the particular catechin so as to obtain a negative finding of " significantly exceeds " as has just been defined. It should also be noted that, in some cases, a finding of " can significantly be justified " with a ratio as small as 2: 1, but more preferably as large as 50: 1 to 100: 1.

In the example of a single catechin compound having an excipient to make up the composition, it may be convenient to merely note and compare the percentage of purity of the compound in the composition, rather than its percentage by overall weight, for purposes of the " significantly " as claimed. In the case of mixtures of catechins, it may be appropriate to consider a combined percent composition of the catechins blended in the therapeutic dosage, and compare that number with a combined percent presence of the same catechins in the natural plant or extract. 40

In any event, the purpose of the disclosed measurement standard is to set a reasonable margin by which a claimed composition exceeds the reading of the natural occurrence of the active ingredients in conventional plants and plant extracts.

Preferred compositions will contain a catechin which is at least substantially pure. It may also advantageously be employed a catechin which is in substantially pure isolated or synthetic form. In general, " pure " means better than 95% pure, and " substantially pure " catechin " means an extraction purified catechin, or other known means or means disclosed herein, so that the catechin is present in the therapeutic dosage only with those impurities which can not be easily or reasonably removed by the extraction or purification processes. " Isolated " means that the catechin in question is not accompanied, in therapeutic form, by significant quantities of other catechins. An " pure " compound " is an isolated purified form compound as is conventional for active ingredients in the pharmaceutical industry.

A Cell Culture Method for Generating Amyloid AA

Some previous studies indicate that AA amyloid formation can be obtained in cell culture model systems (Shirahama et al., Lab. Invest., 62: 61-68, 1990; Palm et al., APMIS 105: 603-608 , 1997; Kluve-Beckerman et al., Am. J. Path, 155: 123-133, 1999). Probably, the best cell culture system where producing fibrillar AA amyloid protein has been described by Kluve-Beckerman et al. (Am. J. Path 155: 123-133, 1999). In this in vitro system, murine peritoneal macrophages (isolated from the peritoneal cavity of normal C57BL / 6 mice) were maintained, to which recombinant SAA2 (rSAA2) was added in neutral pH medium and / or amyloid reinforcing factor (AEF) (discussed down) . The medium containing recombinant SAA2 was used at a concentration typical of that observed in serum in the acute phase (i.e., 140 pg / ml). In a period of 24-48 hours post-treatment, cultures developed foci of AA amyloid deposits, which increased and became increasingly congenital and birefringent (after staining with Congo red, visualized in polarized light). This study demonstrated that SAA2 was cleaved by macrophages to form AA-amyloid protein (~ 8.5 kDa fragment) in culture, which was fibrillar (demonstrated by Congo red birefringence and fluorescence with Thioflavin S positive).

Peritoneal cells were found to be viable (determined by exclusion of trypan blue staining) and maintained phagocytosis activity (shown by latex bead phagocytosis). In addition, cultures of peritoneal cells obtained from nude mice demonstrated similar formation and deposition of amyloid in culture, indicating that T lymphocytes were not essential for the formation of amyloid fibrils.

Although AEF was not required, its addition to the culture medium resulted in larger and more numerous amyloid deposits. A larger amount of fibrillar AA amyloid deposition in culture was also observed when the cultures were treated with pepstatin, an aspartic protease inhibitor (Kluve-Beckerman et al., Am. J. Path., 155: 123-133, 1999). These cultures also maintained a viability similar to that of untreated cultures, even for as long as 24 days. Pepstatin-treated cultures developed large areas of dense and localized amyloid masses associated with cell aggregates.

This cell culture system is easy to employ and prone to manipulation for the development of AA amyloid deposition and persistence in vitro.

A Relevant AA Experimental Amyloidosis Mouse Model

There are some experimental animal models good for the production of systemic AA amyloidosis in vivo. Previous studies involving repeated injections of azocasein (derived from casein) have demonstrated that accumulation of AA amyloid deposits in spleen, liver and kidney occurred within 14-21 days after treatment (Janigan and Druet, Am. J. Path 48: 1013 -1024, 1966). In this model, the injection of azocasein resulted in a sterile abscess which caused an inflammatory reaction, inducing elevated serum levels of SAA in the mouse (Kindy and DeBeer, Methods 9: 701-716, 1999). AA amyloid deposits were fibrillar, indicated by their positive staining with Congo red (ie red / green birefringence, visualized in polarized light).

In more recent animal models, systemic AA amyloid deposition can be induced rapidly in CBA / J mice after a single subcutaneous injection of 2% silver nitrate (the inflammatory stimulus) plus a single intravenous injection of amyloid reinforcing factor (AEF) (discussed below). In this model, amyloid AA deposition occurs first in the spleen (within a period of 24-48 hours), then in the liver (within a 3-4 day period), followed by the kidney (5-7 days) (Axelrad et al. , Am. J. Path, 7: 277-284, 1975;

Kisilevsky et al., Lab. Invest. 33: 544-553, 1977).

We have previously used this model extensively to study the development of AA amyloidosis and the role that specific proteoglycan / glycosaminoglycans play in amyloid formation (Kisilevsky et al., Appl. Path 2: 308-315, 1984; Snow et al. (1987) J. Histochem, Cytochem 39: 1321-1330 (1991)), and the use of the above- . This relevant experimental model is already being used and is specifically used to identify and test the efficacy of potential amyloid AA therapies as described below. The experimental CBA / J mouse model (described below) is an ideal system for testing the efficacy of potential amyloid AA therapies since, after an inflammatory stimulus (ie silver nitrate) and AEF, 100% of the animals will deposit amyloid into tissues within a few days. Although only a small percentage of patients with precursor proteins actually develop amyloid, all animals in this experimental model do so. The inflammatory reaction in CBA / J mice, unlike rheumatoid arthritis or osteomyelitis, for example, leads to the appearance of all prerequisites for the occurrence of amyloid deposition. AEF dramatically shortens the time required for experimental induction of AA amyloid deposition in CBA / J mice and can be demonstrated in tissues 24-48 prior to the amyloid appearance (Axelrad et al., Lab., Invest, 47: 139-146 , 1982). Although the actual identity of the AEF has not been completely resolved, it is believed to be generally protein in nature (Axelrad et al., Lab., Invest. 41: 139-146, 1982), or to consist of a small nest of fibril- (Kisilevsky et al., Amyloid: Int. J. Exp. Clin. Invest., 6: 98-106, 1999). AEF is usually prepared from animal amyloid-filled spleens and / or livers previously induced for accumulation of AA amyloid after daily injections of azocasein or single injections of AEF and silver nitrate. These amyloidotic tissues are homogenized in phosphate buffered saline (PBS) containing protease inhibitors, and the granule is again homogenized a few times. The second to fourth supernatants (obtained after extractions with water) generally contain potent AEF activity, which is used to induce rapidly deposition of AA in animals. The discovery and identification of novel compounds or agents as potential therapeutic agents for halting the formation, deposition, accumulation and / or persistence of amyloid fibrillar amyloid that occur in Alzheimer's disease, systemic AA amyloidosis, type 2 diabetes and other amyloidoses, and fibrillogenesis in Lewy body disease, Parkinson's disease, and multiple system atrophy, are desperately sought after.

Use of Catechins, Bioflavanoids, Flavanols, Flavanodiols, Flavanoids, Tannins (or derivatives of any of these) of Green Tea, Green Tea Leaves, Green Tea Extracts or Green Tea Derivatives, or Other Natural or Synthetic Sources It is known that tea green and other natural sources, such as black tea and wine, contain catechins, bioflavanoids, flavanols, flavanodiols, flavanoids, tannins or derivatives thereof. See Sugita-Konishi et al., &Quot; Epigallocatechin gallate and gallocatechin gallate in green tea catechins inhibit extracellular release of Vero toxin from enterohemorrhagic E. coli ", Biochemica et Biophysica Acta, 1472, 42-50 (1999), and Fernandez et al. ., " HPLC determination of catechins and caffeine in tea ", Analyst 125: 421-425 (2000). We believe now that these constituents play a significant role in the surprisingly beneficial effects of green tea on amyloidoses, as discussed below.

In particular, we believe now that the catechins of the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and / or epicatechin gallate, or a derivative of one of the above group, are effective alone or in combination with other amyloid-inhibiting ingredients, such as any plant matter of the group of plants which consist of and are commonly known as Cat's Claw, gingko biloba, rosemary, gotu kola, bacopin, and ginseng, to obtain any or all of the beneficial effects described in low.

General structures of representative catechins are shown in Figure 1. Pharmaceutically acceptable analogs and derivatives of these catechin structures, such as, for example, various R group type substitutions, and other derivative structural modifications that do not affect the disclosed efficacy of these compounds , can be prepared without affecting the scope of the appended claims. 46

Use of Green Tea Extract Standardized to Inhibit Amyloidosis

All of the Examples illustrated below serve well to establish that, at least in vitro, green tea, green tea leaves and extracts or their derivatives, have the ability to inhibit the formation of amyloid deposits in the brain that occur during normal aging and in a variety of disorders of the brain, including Alzheimer's disease. Additionally, it is known that patients who accumulate amyloid deposits in the brain end up losing cognitive ability and memory function and maintain a marked reduction of overall mental clarity. As a result, it follows that the inhibition of these amyloid deposits in the brain will at least promote a mental alertness in these patients.

The Examples also provide that again, at least in vitro, green tea, green tea leaves and extracts or their derivatives, have the ability to reduce, eliminate, prevent, inhibit, destroy, dissolve, or disintegrate deposits of fibrils or proteins amyloid fibrils, as well as formation of amyloid fibrils, or formation of age-associated amyloid fibrils, and formation of brain-associated amyloid fibrils. In addition, it is known that patients accumulating amyloid fibrillar deposits or amyloid deposits, fibril deposits associated with the brain or amyloid protein deposits associated with the brain, or exhibiting symptoms of formation and growth of amyloid fibrils or formation and growth of amyloid fibrils associated with age, formation and growth of brain-associated amyloid fibrils will usually result in loss of mental acuity, alertness, concentration, cognitive well-being, or some measure of brain function or cognitive ability, mental performance or memory, or concentration and mental alertness, or mental vitality, or mental clarity and alertness, short-term memory, or some of the ability to learn and remember. It is also known that such patients are subject to cognitive or memory decline associated with or related to age, or will maintain a marked reduction of mental clarity.

The inhibition, reduction, elimination, prevention, destruction, disintegration or disintegration of such deposits of amyloid fibrils or proteins, amyloid fibrillar deposits associated with the brain or amyloid protein deposits associated with the brain, or formation and growth of amyloid fibrils , or formation and growth of age-associated amyloid fibrils, will improve mental acuity, promote mental alertness, provide nutritional support for cognitive decline or age-related memory, promote cognitive well-being, support brain function , improve cognitive ability, mental performance or memory, promote concentration and mental acuity, improve mental vitality, promote greater mental clarity and alertness, improve short-term memory, reduce or reverse the cognitive or memory decline associated with age, support normal brain function, intensify learning or memory; improve concentration, enhance mental performance, reduce mental decline, reduce the likelihood of occurrence of age-related brain disorders, and maintain good mental health in these patients. 48

The Examples also suggest that green tea, green tea leaves and extracts, or derivatives thereof, should have the ability to reduce, eliminate, prevent, inhibit, destroy, dissolve, dismantle, disintegrate deposits of fibrils or amyloid proteins, fibril deposits or amyloid proteins associated with the pancreas, as well as formation and growth of amyloid fibrils, and formation and growth of amyloid fibrils associated with the pancreas. Additionally, it is known that patients who accumulate deposits of fibrils or amyloid proteins, fibril deposits or amyloid proteins associated with the pancreas, or who exhibit symptoms of formation and growth of amyloid fibrils, formation and growth of pancreatic associated amyloid fibrils, generally lose a healthy pancreatic function or maintain a reduction of normal insulin function, leading to loss or reduction of pancreatic function. Accordingly, it follows that the inhibition, reduction, elimination, prevention, destruction, disintegration, dissolution or disintegration of such deposits of fibrils or amyloid proteins, deposits of fibrils or amyloid proteins associated with the pancreas, or formation and growth of amyloid fibrils, formation and growth of pancreatic amyloid fibrils will support healthy pancreatic function and promote pancreatic function by helping to promote normal insulin function in these patients.

Recent studies, published after the priority date of this application, now support these experimental results. See Hasegawa, " Preventive effect of Japanese green tea against cognitive impairment in the elderly ", posters 42 and 755, Proceedings of World Alzheimer Congress 2000, in Neurobiology of Aging, 21: 18 (2000), reporting 49 a statistically significant relationship increased green tea beverage and higher cognitive levels.

EXAMPLES

The following examples are provided in order to provide practitioners with the disclosure and description and use of commercially available green tea extract and catechins which surprisingly result in inhibition, dismantling / destruction and / or breakdown of β-containing fibrils in Alzheimer's disease, and NAC fibrils in Parkinson's disease. However, it should not be considered that the invention is limited to these specific examples.

Example 1

Standardized Green Tea Leaf Extract is a Potent Inhibitor of Amyloid Fibrillation Αβ (1-40) in

Alzheimer's disease

A previously described method of measuring the formation of amyloid fibrils using Thioflavin T fluorometry (H Naiki et al., Lab. Invest. 65: 104-110, 1991; H Levine III, Protein Sci: 2: 404-410, 1993; H Levine III, Amyloid:

Int. J. Exp. Clin. Invest. 2: 1-6, 1995; H Naiki and K.

Nakakuki, Lab. Invest. IA, 374-383, 1996) was initially employed to identify whether standardized green tea leaf extract was capable of inhibiting the formation of β-amyloid fibrils in Alzheimer's disease. Using this sensitive assay it has previously been shown that any decreases or increases in fluorescence are correlated with a decrease or increase in the amount of amyloid fibrils (H Naiki et al., Lab. Invest., 65: 104-110, 1991; H Levine III, Protein Sci., 2: 404-410, 1993; H Levine III, Amyloid:

Int. J. Exp. Clin. Invest. 2: 1-6, 1995; H Naiki and K.

Nakakuki, Lab. Invest. 74: 374-383, 1996), allowing the determination of the effects of potential inhibitors and / or enhancers of amyloid fibril formation.

In a first study, the effects of standardized green tea extract as potent inhibitor of amyloid in Alzheimer's disease in the formation of ßβ (1-40) fibrils in Alzheimer's disease were evaluated by Thioflavin T fluorometry. It is known that Thioflavin T binds to fibrillar amyloid proteins, and an increase in fluorescence is correlated with an increased formation of amyloid fibrils, whereas a decrease in fluorescence is correlated with a decrease in the formation of amyloid fibrils. Β-Protein (1-40) in Alzheimer's disease, when incubated at 37 ° C, tends to spontaneously form amyloid fibrils whose amount increases over time. In this study, we tested standardized green tea extract to inhibit the formation of fibrils by amyloid ßβ protein in Alzheimer's disease during a

period of 1 week. For this study, 25 μΜ of ßβ (1-40) (Bachem Inc., Torrance, CA, USA; Lot # WM365) was incubated in microcentrifuge tubes at 37 ° C for 1 week (in triplicate), alone or in the presence 10æg / mL, 50æg / mL or 100æg / mL standardized green tea extract in 150 mM Tris HCl, 10 mM NaCl, pH 7.0 (TBS). For this study, the powder of a standardized green tea extract gelatin capsule obtained from a commercial source (Nature's Resource, Mission Hills, CA) was extracted into 1 mL of distilled water and granulated using a microcentrifuge (for 10 minutes at 2500X g). The supernatant was then collected and lyophilized. A working solution at 1 mg / mL was then prepared for use in the in vitro assays described below using distilled water. Extracts of commercial green tea leaves are usually standardized to 50% polyphenols.

To evaluate the effects of standardized green tea extract on the formation of ßβ (1-40) fibrils, 50 æl aliquots were collected from each tube for analysis at 1 hour, 1 day, 3 days and 1 week. For each determination described above, after each incubation period, 50æl of standardized Αβ +/- standard green tea extract were added to 1.2mL of 100æM Thioflavin T (Sigma Chemical Co., St. Louis, MO) at 50 mM NaP04 (pH 6.0). Studies indicated that increasing concentrations of fibrillar ßβ caused a proportional increase of fluorescence in the presence of 100 æM Thioflavin T, thereby excluding the presence of any disproportionate internal filter effects in these studies. Fluorescence emission at 482 nm was measured on a model 450 Turner Instrument fluorometer at an excitation wavelength of 450 nm. For each determination, the fluorometer was calibrated by setting the zero in the presence of the Thioflavin T reagent alone, and attaching the 50 ng / mL of riboflavin (Sigma Chemical Co., St. Louis, Mo) to the Thioflavin T reagent in 1800 units of fluorescence. All fluorescence determinations were based on these references, and any fluorescence exhibited by any of the compounds in the presence of the Thioflavin T reagent was always subtracted from all relevant readings.

For all fibrillogenesis studies using Thioflavin T fluorometry as disclosed herein, comparisons of amyloid protein in the presence or absence of standardized green tea extract were based on paired Student's t-tests with data presented as a mean + /- standard deviation. Significance was reported at 95% confidence levels (p <0.05), 99% (p <0.01) and 99.5% (p <0.005).

As shown in Figure 2, the effects of standardized green tea extract on γβ (1-40) amyloid fibril formation in Alzheimer's disease were evaluated over a 1-week incubation period. Αβ (1-40) alone, after an incubation of 1 hour at 37øC, demonstrated an initial fluorescence of 183 +/- 10 fluorescence units. During the 1-week incubation period there was a gradual increase of Αβ (1-40) fluorescence alone, increasing approximately 4-fold from 1 hour to 1 day, with a peak fluorescence of 852 +/- 3 fluorescence units observed in the day 1 (Figure 2), consistent with previous studies (Castillo et al., J. Neurochem, 6: 2452-2465, 1997). Standardized green tea extract significantly inhibited γβ (1-40) amyloid fibril formation in a dose-dependent manner as early as 1 hour of incubation (Figure 2). Significant (p <0.005) inhibition of standardized green tea leaf extract from γβ 1-40 amyloid fibril formation was observed at all instants, including 1 hour, 1 day, 3 days, and 1 week (Figure 2 ). At 1 hour, 10æg / mL, 50æg / mL and 100æg / mL of standardized green tea leaf extract significantly (p &lt; 0.005) inhibited the formation of β-1-40 fibrils in 50.3 +/- 3.3%, 66.1 +/- 3.3% and 95.1 +/- 1.6%, respectively. At day 1, 10 æg / mL, 50 æg / mL and 100 æg / mL standardized green tea leaf extract significantly (p &lt; 0.005) inhibited β-1-40 fibrils formation in 36.7 +/- 2.0%, 90.7 +/- 1.0% and 95.9 +/- 2.0%, respectively. At 1 week, 10æg / mL, 50æg / mL and 100æg / mL standardized green tea leaf extract 53 significantly (p &lt; 0.005) inhibited the formation of β-1-40 fibrils at 58.9 + / - 10.7%, 83.0 +/- 1.8% and 89.3 +/- 2.1%, respectively. These initial data indicated that standardized green tea extract and at least one of its active constituents of catechins, bioflavanoids, flavanols, flavanodiols, flavanoids or tannins were potent inhibitors of the formation of amyloid fibrils in Alzheimer's disease.

Example 2

Dismantling / Destruction of Fibrils Amyloid Αβ 1-42 in Alzheimer's Disease by Green Tea Leaf Extract

Standardized

In the following study, standardized green tea leaf extract was tested for its ability to cause a disruption / destruction of preformed amyloid fibrils in Alzheimer's disease containing ßβ 1-42. This type of activity is important for any potential anti-amyloid compound that can be used in patients who already exhibit substantial deposition of amyloid in organs and / or tissues. For example, patients with Alzheimer's disease with intermediate-late-stage disease have abundant Αβ-containing amyloid deposits in their brains as part of neuritic plaques and cerebrovascular amyloid deposits. A compound capable of causing disruption / destruction of pre-existing amyloid deposits will be advantageous for use in these patients who are in later stages of the disease process.

For this study, 1 mg of β-1-42 (Bachem Inc., Torrance, CA, USA; Lot # 516817) was dissolved in 1.0 mL of doubly distilled water (1 mg / mL solution). Then, 25 μΜ of β-1-42 was incubated overnight (-18 hours) at 37 ° C, 54 in the absence or presence of 10 μg / mL, 100 μg / mL or 200 μg / mL leaf extract of standardized green tea in the presence of 150 mM Tris HCl, 10 mM NaCl (pH 7.0) with 0.02% sodium azide. In these studies, (see results described below and in Figure 3), the weight ratio Αβ1-42: green tea extract was 1: 0, 1: 1 and 1: 2, respectively.

For this study, the powder of a standardized green tea extract gelatin capsule obtained from a commercial source (Nature's Resource, Mission Hills, CA) was extracted into 1 mL of distilled water and granulated using a microcentrifuge (for 10 minutes to 2500X g). The supernatant was then collected and lyophilized. A working solution at 1 mg / mL was then prepared for use in the in vitro assays described below using distilled water. Commercial green tea leaf extracts are usually standardized for 50% polyphenols.

A previously described method of measuring the formation of amyloid fibrils using Thioflavin T fluorometry (H Naiki et al., Lab. Invest., 5: 104-110, 1991; H Levine III, Protein Sci., 2: 404-410, 1993, H Levine III, Amyloid: Int. J. Exp. Clin Invest 2: 1-6, 1995; H Naiki and K. Nakakuki, Lab. Standardized green tea leaf extract is capable of causing a dismantling / destruction of γβ 1-42 amyloid fibrils in Alzheimer's disease. It is known that Thioflavin T binds to fibrillar amyloid proteins, and an increase in fluorescence is correlated with an increase in the formation of amyloid fibrils, whereas a decrease in fluorescence is correlated with a decrease in amyloid fibrils due to dismantling 55 and / or destruction. Β-Protein (1-42) in Alzheimer's disease, when placed in solution as distilled water, tends to spontaneously form amyloid fibrils. Using this sensitive assay it has previously been shown that any decreases or increases in fluorescence are correlated with a decrease or increase in the amount of amyloid fibrils (H Naiki et al., Lab. Invest 65: 104-110, 1991; H Levine III, Protein Sci., 2: 404-410, 1993; H Levine III, Amyloid: Int. J. Exp. Clin Invest 2: 1-6, 1995; H Naiki and K. Nakakuki, Lab. Invest 74: 374-383 , 1996), allowing the identification and quantification of the extent of potential inhibitors and / or enhancers of β-1-42 amyloid fibrils in Alzheimer's disease.

To evaluate the effects of standardized green tea extract on the potential disruption / destruction of preformed ßβ 1-42 fibrils, 50 æl ßβ 1-42 +/- standardized green tea leaf extract at various concentrations (described above ), were added to 1.2 mL of 100 μM Thioflavin T (Sigma Chemical Co., St. Louis, MO) in 50 mM NaPO4 (pH 6.0) for fluorometric readings (as described in Example 1).

As shown in Figure 3, increasing amounts of standardized green tea extract caused a dose-dependent dismantling / destruction of preformed ß-1-42 fibrils in Alzheimer's disease. Ssβ 1-42 alone showed an average fluorescence of 780 +/- 50 fluorescence units (Figure 3). Standardized green tea extract at 10æg / ml caused significantly (p &lt; 0.05) a γβ 1-42 fibrillar dismantling / destruction in 17 + 7%. On the other hand, 100 pg / ml and 200 pg / ml caused significantly (p &lt; 0.005) a fibrin dismantling / destruction of β-1-42 in 76 +/- 1.0% and 85 +/- 56 %, respectively. This study demonstrated that standardized green tea extract and at least one of its active constituents of catechins, bioflavanoids, flavanols, flavanodiols, flavanoids or tannins caused disruption / destruction of preformed β-1-42 amyloid fibrils and were effective in a manner dose-dependent.

Example 3

Disaggregation of ßβ 1-42 Fibrils in Alzheimer's Disease by Standardized Green Tea Leaf Extract

In the following study, a spectrophotometric assay of Αβ with Congo red (Klunk et al., Anal. Biochem 266: 66-76, 1999) was modified to determine the efficacy of standardized green tea leaf extract on the breakdown of amyloid fibrils of ß-1-42 in Alzheimer's disease. For this assay, 25 μΜ of ßβ 1-42 (Bachem Inc., Torrance CA, Lot # 516817) was incubated in triplicate for 4 days in distilled water at 37øC in the absence or presence of 400æg / mL extract of standardized green tea (obtained from two commercial sources) in Tris buffered saline (TBS) (100 mM Tris, 50 mM NaCl, pH 7.0, 0.02% sodium azide). The weight ratio Αβ: green tea extract was 1: 4. Source 1 of the standardized green tea extract used in this study came from Sundown Herbals (manufactured and distributed to Sundown Vitamins, Boca Raton, Fl), while source 2 of the standardized green tea extract used in this study came from Nature's Resource (Mission Hills, CA). The green tea used in this study was extracted into distilled water as described in Examples 1 and 2.

After incubation of β-1-42 in the presence or absence of standardized green tea extracts (as described above) 57 (standard green tea extracts from the 2 sources are reported as test compounds), 50 μl of Congo red (Sigma Chemical Co., St. Louis, MO, USA) in distilled water were then added to 250 μl of each incubation mixture, yielding a final molar ratio of Congo's Αβ: red of 1: 3. After 10 minutes, the absorbance at 405 nm (reference wavelength, to account for Congo red absorbance alone at 540 nm) and 540 nm (absorbance of the sample, where &quot; sample &quot; refers to β isolated, test compound alone, or Αβ + test compound, all in the presence of Congo red) using a Biorad Model 550 ELISA Plate Reader (Biorad, Hercules, CA, USA). The absorbance at wavelength of 405 nm was automatically subtracted by the ELISA plate reader from wavelength absorbance at 540 nm (the difference is referred to as Δabsorbance) (Klunk et al., Anal. Biochem 266: 66-76, 1999). As a result, the Δ absorbance reading at 540 nm was proportional to the amount of aggregated Αβ remaining in solution (Klunk et al., Anal. Biochem 266: 66-76, 1999).

For all experiments involving test compounds, reading the absorbance at 540 nm of the test compound alone (in the absence of β) was always subtracted from the absorbance reading corresponding to 540 nm of the test compound in the presence of β.

Using this modification of the method of Klunk et al. (Anal. Biochem 266: 66-76, 1999), the use of a higher final concentration of Congo red (i.e., 60 μ instead of 14 μ) (Anal. Biochem 2, 66: 66-76, 1999 ) in the presence of γβ fibrillar gave an overall absorbance at 540 nm which was always less than 1.0 Absorbance Units (AU), and well within the linear absorbance range.

Standardized green tea leaf extracts from two commercial sources were tested using the Congo red Αβ spectrophotometric assay described above to determine their efficacy in the breakdown of Αβ 1-42 amyloid fibrils in Alzheimer's disease.

As shown in Figure 4, both commercially available standardized green tea extracts caused a breakdown of pre-aggregated β-1-42 amyloid fibrils, determined using the Congo red spectrophotometric assay described above. Ssβ 1-40 alone showed a mean Δ absorbance of 0.141 +/- 0.013 AU (Figure 4). Standardized green tea extract from source 1 (Sundown Herbals) caused a significant (p &lt; 0.005) breakdown of 52.5 +/- 8.5% of 25 μΑ Αβ 1-42 fibrils when incubated at a weight ratio of Αβ l-42: green tea extract of 1: 4 (Figure 4). On the other hand, the standardized green tea extract from source 2 (Nature's Resource) caused a significant (p &lt; 0.005) breakdown of 63.8 +/- 4.2% of 25 μl β-1-42 fibrils when incubated at a weight ratio of Αβ 1-42: green tea extract 1: 4 (Figure 4). Thus, irrespective of the source, standardized green tea leaf extract and at least one of its active constituents of catechins, bioflavanoids, flavanols, flavanodiols, flavanoids or tannins were potent disintegrators of Αβ1-42 amyloid fibrils in Alzheimer's disease. 59

Example 4

Catechesins are Potent Inhibitors of the Formation of Fibrils Amyloid of ßβ (1-40) in Alzheimer's Disease

A previously described method of measuring the formation of amyloid fibrils using Thioflavin T fluorometry (H Naiki et al., Laboratory U5: 104-110, 1991; H Levine III, Protein Sci: 2: 404-410, 1993; H Levine III, Amyloid: Int. J. Exp. Clin. Invest. 2: 1-6, 1995; H Naiki and K. Nakakuki, Lab. Invest. 74: 374-383, 1996) was initially employed to identify whether extract of standardized green tea leaves was able to inhibit the formation of β-amyloid fibrils in Alzheimer's disease.

In a first study the effects of commercially available catechins as a potent inhibitor of amyloid in Alzheimer's disease in the formation of ßβ (1-40) fibrils in Alzheimer's disease by Thioflavin T fluorometry were evaluated. Β- 40) in Alzheimer's disease, when incubated at 37 ° C, tends to spontaneously form amyloid fibrils whose amount increases over time. In this study we tested the ability of catechins to inhibit the formation of fibrils by the amyloid β protein in Alzheimer's disease over a period of 1 week. For this study, 125 μΜ of ßβ (1-40) (Bachem Inc., Torrance, CA, USA) was incubated in microcentrifuge tubes at 37 ° C for 1 week (in triplicate), alone or in the presence of catechins of different commercial sources including Epicatechin (ICN Pharmaceuticals Inc., Costa Mesa, CA), Epicatechin (Aldrich, St. Louis, MO), Hydrated Catechin (Fluka Chemica-Biochemika, Ronkonkoma, New York) and Catechin (Aldrich, St. Louis, MO) in 50 mM Tris HCl, 50 mM NaCl, pH 7.0 (TBS). Increasing amounts of catechins (as described above) were assayed including 60 Aβ: catechin at a weight-to-weight ratio of 1: 1, 1: 0.1, 1: 0.01 and 1: 0.001 to determine whether the catechins exerted a dose-dependent inhibition of γβ 1-40 fibrils formation.

To evaluate the effects of catechins on the formation of ßβ (1-40) fibrils, aliquots were collected from each tube for analysis at 0, 3, and 7 days. For each determination described above, after each incubation period, 10æl of Αβ +/- catechins were added to 1.2mL of 100μΜ Thioflavin T (Sigma Chemical Co., St. Louis, MO) in 50mM NaP04 (pH 6.0). Studies indicated that increasing concentrations of fibrillar ßβ resulted in a proportional increase of fluorescence in the presence of 100 μg Thioflavin T, thereby excluding the presence of any disproportionate internal filter effects in these studies. Fluorescence emission at 482 nm was measured on a model 450 Turner Instrument fluorometer at an excitation wavelength of 450 nm. For each determination, the fluorometer was calibrated by setting the zero in the presence of the Thioflavin T reagent alone, and attaching the 50 ng / mL of riboflavin (Sigma Chemical Co., St. Louis, Mo) to the Thioflavin T reagent in 1800 units of fluorescence. All fluorescence determinations were based on these references, and any fluorescence exhibited by any of the compounds in the presence of the Thioflavin T reagent was always subtracted from all readings.

For all fibrillogenesis studies using Thioflavin T fluorometry, as disclosed herein, comparisons of amyloid protein in the presence or absence of catechins were based on paired Student t-tests, with data presented as mean +/- standard deviation. Significance was reported at 95% confidence levels (p <0.05), 99% (p <0.01) and 99.5% (p <0.005).

The effects of commercially available catechins on the formation of β-amyloid fibrils (1-40) in Alzheimer's disease were evaluated over a 1-week incubation period. During the 1-week incubation period a Αβ (1-40) fluorescence gradual increase occurred alone, with a peak fluorescence of 450 +/- 46 fluorescence units observed at 7 days (Figure 5), consistent with previous studies (Castillo et al., J. Neurochem 6: 2452-2465, 1997). All of the epicatechin (from ICN and Aldrich), catechin hydrate and catechin inhibited the formation of Αβ (1-40) amyloid fibrils in a dose-dependent manner, and significantly (p &lt; 0.01) at weight ratios of Aβ: catechin 1: 1 and 1: 0.1 (Figure 5). A 1: 1 weight / weight ratio of Αβ: epicatechin (ICN) inhibited the formation of Αβ fibrils by 85%, whereas a weight / weight ratio of 1: 0.1 Αβ: epicatechin (ICN) inhibited γβ fibrillation formation by 67%. A 1: 1 weight / weight ratio of Αβ: epicatechin (Aldrich) inhibited the formation of Αβ fibrils by 86%, while a weight / weight ratio of 1: 0.1 Αβ: epicatechin (ICN) inhibited formation of Αβ fibrils by 62%. A 1: 1 weight / weight ratio of Αβ: catechin hydrate (Aldrich) inhibited the formation of Αβ fibrils by 88%, whereas a weight / weight ratio of 1: 0.1 Αβ: catechin (Aldrich) inhibited the formation of Αβ fibrils by 62%. A 1: 1 weight / weight ratio of ß: catechin (Aldrich) inhibited the formation of ßβ fibrils by 85%, whereas a weight / weight ratio of 1: 0.1 Å: catechin (Aldrich) inhibited fibrillar formation of 66%. These initial data indicated that catechins, including epicatechin, catechin hydrate and catechin, were potent inhibitors of the formation of amyloid fibrils in Alzheimer's disease.

Example 5

Inhibition of Fibrillation Formation of β-Amyloid by Catechins Demonstrated by a Reduction of Congo Red-birefringence Inhibition of β-1-40 fibrillation by catechins was confirmed by a loss of birefringence with Congo red as demonstrated in staining assays with Congo red (Figure 6). In these studies, 125 μΜ μl aliquots alone, or Αβ + catechins were incubated for 1 week at 37 ° C, and then placed on gelatin coated slides, air dried overnight and stained with Congo red. A marked reduction of birefringence with Congo red (visualized in polarized light) in the presence of epicatechin (ICN) (Figure 6) was shown (Figure 6) at a weight ratio of Αβ: epicatechin of 1: 0.5 (Fig. 6B) and, to a lesser extent, at a weight ratio of β: epicatechin of 1: 0.05 (Fig. 6C). Figure 6A demonstrates Congo red staining of Αβ amyloid deposits after incubation of 125 μΜ of Αβ 1-40 at 37 ° C for 1 week alone. This study demonstrated that catechins, such as epicatechin, were potent inhibitors of the formation of β-amyloid fibrils.

Example 6

Dismantling / Destruction of Fibrils Amyloid of ß-1-42 in Alzheimer's Disease by Catechins

In the following study, catechins available on the market were tested for their ability to cause a disruption / destruction of pre-63 amyloid fibrils formed in Alzheimer's disease containing ßβ 1-42. This type of activity is important for any potential anti-amyloid compound that can be used in patients who already exhibit substantial deposition of amyloid in organs and / or tissues. For example, patients with Alzheimer's disease with intermediate-late-stage disease have abundant Αβ-containing amyloid deposits in their brains as part of neuritic plaques and cerebrovascular amyloid deposits. A compound capable of causing disruption / destruction of pre-existing amyloid deposits will be advantageous for use in these patients who are in later stages of the disease process.

For this study, 1 mg of β-1-42 (Bachem Inc., Torrance, CA, USA) was dissolved in 1.0 mL of doubly distilled water (1 mg / mL solution). Then 25 μΜ of ßβ 1-42 was incubated at 37øC in the absence or presence of epicatechin (from Fluka), epicatechin (from ICN), hydrated catechin (from Aldrich), galhocatechin gallate (from Sigma), Galhato (from Sigma), Epicatechin (from Sigma), Epicatechin from green tea (from Sigma) in the presence of 150 mM Tris HCl, 10 mM NaCl (pH 7.0) with 0.02% sodium azide. In these studies (see results described below and in Figure 7) the weight ratios of ßβ 1-42: catechin of 1: 1, 1: 0.1 and 1: 0.01 were tested. The Thioflavin T fluorometry assay was used as described in Example 1.

As shown in Figure 7, increasing amounts of commercially available catechins have caused a dose-dependent dismantling / destruction of preformed ßβ 1-42 fibrils in Alzheimer's disease. Β-1-42 alone demonstrated an average fluorescence of 1037 +/- 641 units of fluorescence (Figure 7). All of the epicatechin (from Fluka, ICN, Aldrich and Sigma), hydrated catechin, galhocatechin gallate and epicatechin gallate caused a destruction / dismantling of preformed Αβ (1-42) amyloid fibrils in a dose-dependent manner, and significantly (p &lt; 0.01) at Aβ: catechin weight ratios of 1: 1 and 1: 0.1 (Figure 6). A 1: 1 weight / weight ratio of ß: epicatechin (Fluka) destroyed fibrin formation of β-1-42 in 86%, whereas a weight / weight of 1: 0.1 of β-epicatechin (Fluka) destroyed β-1-42 fibrillar formation in 63%. A 1: 1 weight / weight ratio of ß: epicatechin (ICN) destroyed fibrillar formation of ß-1-42 in 91%, while a 1: 0.1 weight: æl: epicatechin (ICN) weight / destroyed fibrillar formation of β-1-42 in 78%. A 1: 1 weight / weight ratio of ß: epicatechin (Aldrich) destroyed the fibrillar formation of ß-1-42 in 85%, whereas a 1: 0.1 weight: æl: epicatechin (Aldrich) destroyed fibrillar formation of β-1-42 in 74%. A 1: 1 weight / weight ratio of Αβ: hydrated catechin (Aldrich) destroyed Αβ 1-42 fibrillar formation by 86%, whereas a weight / weight ratio of 1: 0.1 Αβ: hydrated catechin ( Aldrich) destroyed fibrillar formation of β-1-42 in 60%. A 1: 1 weight / weight ratio of Αβ: galhocatechin gallate (Sigma) destroyed Αβ 1-42 fibrillar formation by 86%, while a weight / weight ratio of 1: 0.1 Αβ: ρ3ΐ1ΐ3Ρο galhocatechin (Sigma) destroyed fibrillar formation of β-1-42 in 44%. A 1: 1 weight / weight ratio of Αβ: epicatechin gallate (Sigma) destroyed β-1-42 fibrillar formation by 92%, while a weight / weight ratio of 1: 0.1 Αβ: β-galactate epicatechin (Sigma) destroyed β-1-42 fibrillar formation in 65%. A 1: 1 weight / weight ratio of Αβ: epicatechin (Sigma) destroyed fibrillar formation of Αβ 1-42 in 85%, while a weight / weight ratio of 1: 0.1 Αβ: epicatechin (Sigma ) destroyed fibrillar formation of β-1-42 in 81%. A 1: 1 weight / weight ratio of Αβ: green tea epicatechin (Sigma) destroyed Αβ 1-42 fibril formation by 86%, whereas a weight / weight ratio of 1: 0.1 Αβ: epicatechin of green tea (Sigma) destroyed β-1-42 fibrillar formation in 63%. This study demonstrated that all catechins, including epicatechin (from Fluka, ICN, Aldrich and Sigma), hydrated catechin (Aldrich), galhocatechin gallate (Sigma) and epicatechin gallate caused a dismantling / destruction of amyloid fibrils of ß- 42 and were effective in a dose-dependent manner.

Example 7

Catechins are Potent Inhibitors of Fibrillar Formation in Parkinson's Disease

In the following study, commercially available catechins were tested for their ability to inhibit the formation of alpha-synuclein fibrils in Parkinson's disease. NAC (also known and sometimes referred to herein as NAC-P) was used, a 35 amino acid fragment of alpha-synuclein, since it is known to spontaneously form amyloid-type fibrils after incubation at 37 ° C. For this study, 62.5 μl of NAC (Bachem Inc., Torrance, CA, USA) was incubated in microcentrifuge tubes at 37 ° C for 1 week (in triplicate) alone or in the presence of catechins from different commercial sources, including Epicatechin (EC) (Fluka), Epicatechin (EC) (ICN), Epicatechin (EC) (Aldrich), Epicatechin (EC) (Sigma), Epicatechin gallate (Sigma), Hydrated catechin (Aldrich) and Galhocatechin gallate Sigma) in 150 mM Tris HCl, 10 mM NaCl, pH 7.0 (TBS). Increasing amounts of catechins (as described above) were tested including NAC: catechin at a weight ratio of 1: 1, 1: 0.5, 1: 0.1, and 1: 0.01 to determine if the catechins exerted a dose-dependent inhibition of the formation of NAC fibrils. To evaluate the effects of catechins on the formation of NAC fibrils, aliquots from each tube were assayed at 0, 3, and 7 days using the Thioflavin T fluorometry procedure as described in Example 4.

The effects of commercially available catechins on the formation of NAC fibrils were evaluated over a 1-week incubation period. During the 1-week incubation period a gradual increase of NAC fluorescence occurred, with a peak fluorescence of 381 +/- 50 fluorescence units observed at 7 days (Figure 8). All of the epicatechin (from Fluka, ICN, Aldrich and Sigma), epicatechin gallate (Sigma), hydrated catechin (Aldrich) and galhocatechin gallate (Sigma) inhibited the formation of NAC fibrils in a dose-dependent manner, and significantly (p <0.01) at weight ratios of NAC: catechin 1: 1, 1: 0.5, 1: 0.1 and 1: 0.01 (Figure 8). A weight / weight ratio of 1: 0.1 NAC: epicatechin (Fluka) inhibited the formation of NAC fibrils by 70%, whereas a weight / weight ratio of 1: 0.01 NAC: epicatechin (Fluka) inhibited the formation of NAC fibrils by 46%. A 1: 0.1 weight / weight ratio of NAC: epicatechin (ICN) inhibited the formation of NAC fibrils by 89%, whereas a weight / weight ratio of 1: 0.01 NAC: epicatechin (ICN) inhibited the formation of NAC fibrils by 49%. A weight / weight ratio of 1: 0.1 NAC: epicatechin (Aldrich) inhibited the formation of NAC fibrils by 76%, whereas a weight / weight ratio of 1: 0.01 NAC: epicatechin (Aldrich) inhibited the formation of 67 NAC fibrils in 33%. A weight / weight ratio of 1: 0.1 NAC: epicatechin (Sigma) inhibited the formation of NAC fibrils by 75%, whereas a weight / weight ratio of 1: 0.01 NAC: epicatechin (Sigma) inhibited the formation of NAC fibrils by 40%. A weight / weight ratio of 1: 0.1 NAC: epicatechin gallate (Sigma) inhibited the formation of NAC fibrils by 77%, while a weight / weight ratio of 1: 0.01 NAC: epicatechin (Sigma) inhibited the formation of 34% NAC fibrils. A 1: 0.1 weight / weight ratio of NAC: hydrated catechin (Aldrich) inhibited the formation of NAC fibrils by 77%, whereas a weight / weight ratio of 1: 0.01 NAC: catechin hydrate ( Aldrich) inhibited the formation of NAC fibrils by 34%. A 1: 0.1 weight / weight ratio of NAC: galhocatechin gallate (Sigma) inhibited the formation of NAC fibrils by 73%, whereas a weight / weight ratio of 1: 0.01 NAC: galhocatechin (Sigma) inhibited the formation of 34% NAC fibrils. This study demonstrated that all catechins, including epicatechin (from Fluka, ICN, Aldrich and Sigma), hydrated catechin (Aldrich), galhocatechin gallate (Sigma) and epicatechin gallate (Sigma) caused an inhibition of the formation of NAC fibrils and were effective in a dose-dependent manner.

Example 8

Screening and Identification of Specific Polyhydroxylated Aromatic Compounds as AA Amyloidosis Inhibitors Using a Cell Culture Model System

Specific polyhydroxylated aromatic compounds are believed to be potent inhibitors / destructors of the formation and deposition of fibrillar AA amyloid in cell culture. A group of about 30 commercially available polyhydroxylated aromatic compounds which differ in the position of various functional hydroxyl groups is believed to inhibit the formation and deposition of fibrillar AA amyloid using a model cell culture system. A murine macrophage culture system receiving recombinant amyloid A 2 (SAA 2) and amyloid reinforcing factor (AEF) is used to generate fibrillar AA amyloid in vitro. Deposition of fibrillar AA amyloid, detected by Congo red staining and fluorescence with Thioflavin S, is quantitated using an imaging system. Additionally, quantification of AA amyloid formation and deposition is accomplished by analyzing the cell layer and culture derived media using SDS-PAGE, immunostaining and scanning densitometry. Selected effective compounds which are found to inhibit the formation and / or deposition of amyloid AA in culture are then tested in an experimental model of AA amyloidosis mouse.

Example 9

Determination of the Efficacy of Selected Polyhydroxylated Aromatic Compounds in a Systemic Amyloidosis AA Mouse Model

Specific polyhydroxylated aromatic compounds are believed to be potent inhibitors of the formation and deposition of fibrillar AA amyloid in an experimental amyloidosis AA mouse model. The selected effective compounds identified as above are used to orally (gavage) groups of CBA / J mice (n = 8 per group) over a 21 day treatment period. Fibrillary amyloid AA is induced in these animals on day 8 of the treatment period by a single subcutaneous injection of silver nitrate (inflammatory stimulus), followed by a single injection into the tail vein of 100 pg AEF. A group of 12 animals treated orally with phosphate buffered saline (PBS) and induced for AA amyloidosis (at day 8) serves as the control group. Spleen, liver and kidneys are obtained from each animal and the% amyloid load on each organ is quantified using image analysis after Congo red staining or fluorescence with Thioflavin S. In addition, immunohistochemistry is implemented for the detection of amyloid protein AA and its precursor (i.e., SAA), as well as other known components associated with AA amyloid (i.e., heparan sulfate proteoglycans / glycosaminoglycans, amyloid P component, and ApoE), to determine whether reduction of deposition of amyloid AA in tissues is also correlated with a reduction of components associated with amyloid.

We internally established the use of the experimental model of AA mouse. First, AEF was kindly provided by Dr. Robert Kisilevsky (Queen's University, Kingston, Ontario, Canada). Female CBA / J mice were then injected subcutaneously with a single injection of 0.5 ml of 3% silver nitrate (to induce an inflammatory reaction) and a single injection into the tail vein of 100æg AEF. Using this model, within a period of 2-3 days after the injection, abundant AA amyloid deposition occurred in the spleen, followed by liver and kidney. As shown in Figure 9, fibrillar AA amyloid deposits were first observed in the perifollicular areas of the spleen. Congo red staining of tissue sections revealed characteristic red / green birefringence (visualized in polarized light) indicative of fibrillar amyloid deposits (Fig. 9A). The presence of fibrillar amyloid deposits was also confirmed by positive fluorescence with Thioflavin S (Fig. 9C). Confirmation of AA amyloid deposits in spleen, liver and kidney was demonstrated by positive immunostaining using a polyclonal antibody against amyloid AA protein (Serotek) (not shown). In addition, as previously described in some studies (Snow et al., Lab, Invest 5: 37-44, 1985; Snow et al., Lab. Snow et al., J. Histochem., Cytochem., 39: 1321-1330 (1991)). AA amyloid deposits have demonstrated a colocalization of heparan sulfate glycosaminoglycans (probably as part of proteoglycans), shown using a phage-generated periplasmic fraction antibody (known as HS4C3, which recognizes a specific heparan sulfate GAG epitope) van Kuppevelt et al., J. Biol. Chem. 273: 12960-12966, 1998) (Fig. 9C). Figure 9 shows the deposition of amyloid in the spleen in an experimental amyloidosis AA mouse model. The panels represent splenic tissue collected 5 days after CBA / J mice were injected with 100 æg of AEF +0.5 ml of 3% silver nitrate. (Fig. 9A) Splenic amyloid in the perifollicular area (light areas, arrows), shown by Congo red staining (ie red / green birefringence), visualized in polarized light. (Fig. 9B) Heparan sulphate glycosaminoglycans in the splenic perifollicular areas (dark areas, arrows) (Fig. 9B) Splenic amyloid (clear areas, arrows) in the perifollicular area in another animal, demonstrated by positive fluorescence with Thioflavin S. ), demonstrated by immunostaining with an antibody generated by phage display (known as HS4C3), which recognizes a specific heparan sulfate GAG epitope. All figures X100. 71

In the liver, fibrillary AA amyloid deposits were first observed, within 3-4 days, in the walls of the central veins (Fig. 10A), followed by deposition of amyloid throughout the parenchyma by days 5-7 (Fig. 10C). The nature of the fibrillar amyloid deposits was confirmed by Congo red staining (i.e., red / green birefringence visualized in polarized light) (Fig. 10A, 10C) and Thioflavin S (i.e., positive fluorescence) . Figure 10 shows the deposition of amyloid in the liver in an experimental amyloidosis AA mouse model. Panels A and B represent liver tissue harvested 7-10 days after CBA / J mice were injected with 100æg AEF + 0.5ml of 3% silver nitrate. (Fig. 10A) Amyloid in the liver (at 7 days) mostly confined to the walls of a central vein (clear areas, arrows), shown by Congo red staining (ie red / green birefringence), visualized in polarized light (arrows). X100 (Fig. 10C) Amyloid in the liver (at 7 days) in the walls of central veins (light areas, arrows) and penetrating the parenchyma (arrowheads), detected by fluorescence with Thioflavin S. X100 (at 10 days) in the walls of the central veins (clear areas, arrowheads) and throughout the liver parenchyma after 10 days after the injection of AEF + AgN03. X25.

These studies indicate that the experimental model of systemic AA amyloidosis mouse is feasible. We used this model to test the efficacy of AA amyloid inhibitors after initial screening using the cell culture model system.

Example 10

Identification of a New Class of Compounds as Agents

Potential Antiamyloid Most of our previous studies involved the analysis of these compounds for the destruction / dismantling of preformed amyloid fibrils consisting of beta-amyloid protein (seen in amyloid deposits in Alzheimer's disease), islet amyloid polypeptide (seen in the islets of 90% of patients with type 2 diabetes), alpha-synuclein (seen in deposits in Parkinson's disease), or prion protein (seen in prion diseases). Our initial studies demonstrate that specific polyphenolic compounds can cause destruction / dismantling of different types of amyloid proteins and thus tend to be less specific, whereas other polyphenolic compounds tend to destroy only one type of amyloid protein (and thus, are more specific). Below are examples of some of the polyhydroxylated aromatic compounds we have screened for inhibition of formation and deposition of amyloid AA.

As an example, Figure 11 demonstrates the effect of specific polyhydroxylated aromatic compounds on the destruction / dismantling of amyloid fibrils consisting of islet amyloid polypeptide (IAPP) (the amyloid protein that accumulates in pancreatic islets of -90% of patients with diabetes (Westermark, 1972, Clark et al., 1988), evaluated by fluorometry with Thioflavin T (Naiki et al., 1991; Levine III, 1993; In this assay, 73 increases or decreases in Thioflavin T fluorometry are proportional to increases or decreases in the amount of IAPP fibrils (Naiki et al., 1991; Levine III, 1993; As shown in Figure 11, after a 1-week incubation at 37 ° C, 25 μl of IAPP alone demonstrated 5806 +/- 301 fluorescence units, indicating the presence of abundant IAPP amyloid fibrils. All IAPP fibrils were incubated for 1 week at 37 ° C at a IAPP weights ratio: 1: 0.1 compound (approximate molar ratio 1: 1) with specific polyhydroxylated aromatic compounds, including epicatechin (EC; Fluka), epicatechin gallate (ECG, Sigma) or pyrocatechol (Sigma), demonstrated a marked destruction / dismantling of 90-96% of preformed IAPP fibrils. On the other hand, EDTA (as negative control) and the polyphenolic acid known as quinic acid did not cause any significant destruction / dismantling of preformed IAPP fibrils (Fig. 11). Direct destruction of preformed IAPP fibrils by specific polyphenolic compounds was also confirmed by Congo red staining assays (not shown). Figure 11 shows that specific polyhydroxylated aromatic compounds act as potent destroyers of preformed amyloid fibrils. This graph is a fluorometry assay with Thioflavin T for 1 week to identify inhibitors of IAPP fibrils. As shown, only epicatechin (EC), epicatechin gallate (ECG) and pyrocatechol destroy / dismantle preformed IAPP fibrils by 90-96%.

In our other studies circular dichroism spectroscopy is used to test the efficacy of certain polyphenols in the destruction of preformed fibrils consisting of beta-amyloid protein (Αβ) 1-42, a protein that forms amyloid fibrils as part of amyloid plaques in the brains of all patients with Alzheimer's disease. As an example, the effects of the polyphenolic epicatechin on the destruction of β-1-42 fibrils were tested. In this study, 50 μΜ of β-1-42 (Bachem Inc) was incubated in the absence or presence of epicatechin (Sigma) for 1 week at 37 ° C, at a weight / weight ratio of Αβ: ΕΟ of 1: 1 approximately 1:10). As shown in Figure 12, ßβ 1-42 alone demonstrates the typical spectra of a fibrillar amyloid protein containing a structure in pleated β sheets by the tagged minimum observed at 222 nm. On the other hand, incubation with epicatechin for 3 days resulted in significant destruction / dismantling of the β-sheet structure (Fig. 12), suggesting that the mechanism of action of specific polyhydroxylated aromatic compounds may involve destruction of pleated β sheets. Figure 12 shows the epicatechin destruction / dismantling of the pre-formed β-sheet structure of preformed amyloid fibrils in Alzheimer's disease. Circular dichroism spectroscopy was used to evaluate the ability of epicatechin (EC) to cause a destruction / dismantling of the structure in pre-formed β-sheets of preformed β-1-42 fibrils. As shown, at 3 days of coincubation, epicatechin was effective in the destruction of β-sheet structure (revealed by changing the spectra, especially at 222 nm).

Example 11

Inhibition of Amyloid Experimental Amyloidosis AA by

Epicatechin 75

Studies also demonstrate that the specific polyhydroxylated aromatic compound known as epicatechin (C 15 H 14 O 6; MW 290.3) has the ability to markedly prevent the deposition of AA amyloid in the spleen and stable in the mouse model (i.e., after administration of AEF + silver nitrate). This was not observed after treatment with catechin (the epicatechin epimer), suggesting that structure-activity relationships exist for the inhibition of the formation and deposition of AA amyloid fibrils by polyhydroxylated aromatic compounds.

In this study, 4 groups (n = 6 per group) of 8 week old female CBA / J mice were first pretreated for 7 days with a) phosphate buffered saline (PBS), b) epicatechin (C 15 H 14 O 6; 290.3) (Fluka, St. Louis, MO) or c) hydrated catechin (C 15 H 406; MW 290.3) (Fluka, St. Louis, MO). Epicatechin was administered daily by oral gavage (0.5 ml test article in PBS, pH 7.4) at a dosage of 78 mg / kg / day (epicatechin solubility limit), whereas the hydrated catechin was administered at a high dose (500 mg / kg / day).

After a pre-treatment period for 7 days with PBS, epicatechin or catechin, all animals were induced for AA amyloid deposition by a single subcutaneous injection of 0.5 ml silver nitrate (inflammatory stimulus), followed by a single Injection into the tail vein of 100 æg of AEF in distilled water. The mice were then treated for an additional 14 days with PBS, epicatechin or catechin by oral gavage as described above (total treatment time with the test compound 76 days). At the end of the experimental protocol, the animals were sacrificed and the spleen, liver and kidneys were collected for histological analysis.

Sections of tissue (i.e., spleen, liver and kidney) were then stained for fibrillar amyloid using Congo red and Thioflavin S. Five organ sections were then used to assess amyloid burden. In these studies an arbitrary scoring system (from 0 to 5) was used, and the amyloid load per organ was determined by the blind score of two investigators. As an example, for the scoring of tissue sections with Congo red in the spleen, 0 = no Congo red birefringence in tissue; 1 = slight birefringence with Congo red confined to a portion of the perifollicular area; 2 = Congo red birefringence confined to two or more portions of the perifollicular area; 3 = Congo red birefringence occupying the entire perifollicular area; 4 = Congo red birefringence occupying the entire perifollicular area and penetrating the white pulp; 5 = Congo red birefringence occupying the entire perifollicular area and penetrating deeply into the white pulp. We also used a more quantitative approach to determine amyloid% loading in various tissues using a more sophisticated imaging system.

As shown in Figure 13, animals receiving saline alone demonstrated marked deposition of amyloid in the spleen at the end of the 2-week amyloid induction period, with a Congo red score of 3.83 +/- 0.1. Hydrated catechin (i.e., Congo red score of 3.20 +/- 0.46) 77 reduced the amyloid AA deposition in the spleen by 16.5%, but this value was not significant. However, epicatechin (i.e., Congo red score of 2.16 +/- .050) significantly inhibited the deposition of fibrillar AA amyloid in the spleen in 43.7% (Fig. 13). A similar inhibition of fibrillar AA amyloid in the liver and kidney was also observed after treatment with epicatechin, but not with catechin (not shown). The inhibitory effect of epicatechin is so potent that some of the animals even demonstrated little or no fibrillar AA amyloid in tissues after treatment (Figure 14). This study demonstrates that selected polyhydroxylated aromatic compounds have the potential to serve as potential therapies for the treatment of systemic AA amyloidosis. The described studies employ cell culture techniques and an experimental model of AA amyloidosis mouse to identify polyhydroxy aromatic compounds that serve as prototype compounds for the treatment of AA amyloidosis. Figure 14 shows a marked reduction of splenic AA amyloid deposition by epicatechin, revealed by Congo red staining. (Fig. 14A) Congo red staining of AA amyloid deposits in the perifollicular area of the spleen (arrows) in a mouse induced with AEF + AgN03 (day 8), and treated with PBS (for 21 days). Color photomicrographs demonstrate red / green birefringence indicative of fibrillar amyloid (visualized in polarized light). (Fig. 14B) Near-complete elimination of AA amyloid deposits in the perifollicular area of the spleen (arrows), shown by absence of Congo red staining (visualized in polarized light) of an induced animal 78 with AEF + AgNC &gt; 3 (day 8 ), and treated with epicatechin (for 21 days).

Example 12

Identification of New AA Amyloid Inhibitors - Screening

In vitro

Our studies suggest that certain polyhydroxylated aromatic compounds, such as epicatechin, may serve as potent inhibitors of the formation and deposition of fibrillar AA amyloid in tissues. We use novel cell culture techniques to track some known and commercially available polyhydroxylated aromatic compounds that differ in the number of aromatic rings and / or the position of various hydroxyl groups and other functional groups on the aromatic rings. This type of structure-activity relationship (SAR) screening studies illuminates the important structural features necessary for effective inhibition of formation and deposition of AA amyloid. A murine macrophage culture system is utilized using recombinant SAA2 and AEF to generate fibrillar AA amyloid in vitro. Deposition of fibrillar AA amyloid detected by Congo red staining and Thioflavin S fluorescence is quantitated in culture using an imaging system equipped for fluorescence and polarization. In addition, the quantification of amyloid AA formation is obtained by analyzing the cell and media layer using SDS-PAGE, immunostaining and scanning densitometry. When AA amyloid is established in culture, the cultures are screened with selected polyhydroxylated compounds at increasing concentrations. The most effective compounds which are found to inhibit the formation and deposition of amyloid AA in culture are then tested in a relevant experimental model of AA amyloidosis mouse.

Establishment of a Cell Culture System for the Study of AA Amyloidosis

For the establishment of a cell culture system to screen for inhibitors of formation and deposition of fibrillar AA amyloid, we used the method described by Kluve-Beckerman et al. (1999).

Collection and Culture of Macrophage / Murine Peritoneal Cells

Cells are harvested by lavage from the peritoneal cavity of normal 8-10 week old female C57B1 / 6 mice. In summary, 8 ml of collection medium (RPMI 1640; Life Technologies, Grand Island, NY), 25 mM HEPES, pH 7.0 and IX antibiotic-antimycotic (Life Technologies) are injected intraperitoneally, the abdomen is gently massaged , and the fluid and cells are withdrawn. Cells are harvested by centrifugation, resuspended at a concentration of 5 X 10 6 cells / mL and plated at a density of 1.7 X 106 cells / well in 8-well chamber slides. Culture media contains RPMI 1640, 2 mM L-glutamine, antibiotic-antimycotic IX, and 15% fetal calf serum (Hyclone Labs, Logan, VT). Binding of the cells is allowed for 3 hours, after which the cells are extensively rinsed to remove non-adherent cells. Adherent cells exhibit a typical round uniform morphology of freshly plated non-activated macrophages. The cells are maintained in 350 æl of culture medium per well at 37øC in an atmosphere of 5% CO 2, the media being replaced every 2-3 days. After addition of recombinant AEF and SAA2 (as described below) for the induction of AA amyloid deposition in culture (usually observed within 2-4 days of induction), cell cultures are maintained for a period of 14 -20 days.

Preparation of the Amyloid Reinforcement Factor AEF is prepared from the spleens of amyloidotic mice after a treatment for 2 weeks with a single subcutaneous injection of 0.5 ml of AgNCb and a single injection in the tail vein of 100æg of AEF in water distilled. The initial AEF material was kindly provided by Dr. Robert Kisilevsky (Queen's University, Canada), and more AEF was generated using the method of Pras et al. (1968). In summary, approximately 2 grams (10 amyloidotic spleens) of tissue are processed for each preparation. The spleens are homogenized using a Kontes fabric shredder in 40 mL of 0.15 M NaCl salt solution until the tissue is in the form of a thin paste. The slurry is then centrifuged in a bench microfuge at 10,000 rpm for 30 minutes. The supernatant is wasted and the pellet resuspended in 40 mL of saline and centrifuged again as described above. This process is repeated seven times. The salt is then removed from the granules by further homogenization in sterile distilled water and centrifugation in a bench microfuge at 15,000 rpm for 2 hours. The supernatant is wasted and the pellet resuspended in 20 mL of distilled water and centrifuged again at 15,000 rpm for 2 hours. This time, the supernatant is not wasted and is designated Supernatant II (Sob II), which contains significant AEF activity. The process used for the generation of Sob II is repeated, and the supernatant, designated Under III, is stored. Granule 81 obtained from the last step is again suspended in 7 ml of distilled water and centrifuged at 10,000 rpm for 3 hours. The supernatant is stored and is designated Under IV. Under II, Under III and Under IV contain AEF activity, which is evaluated by the method of Bradford (Bradford, 1976) to determine the protein concentration for each supernatant. The 3 supernatants are then pooled and lyophilized in order to obtain multiple 100 μg lyophilized aliquots. AEF is used for cell culture (target 1) studies and induction of experimental AA amyloid in a mouse model (objective 2), as described below. We have found that 10 amyloidotic spleens give approximately 4 mg of AEF (sufficient for injections to 40 animals, i.e. 100 Âμg per animal as described herein.

Recombinant SAA2 mouse SAA2, corresponding to SAA in CE / J strain (EC / J SAA), is produced in Escherichia coli BL834 cells using the pET-21a vector as previously described (Kluve-Beckerman et al., 1997). Purification from E. coli lysates is accompanied by chromatography with Sepharose CL-6B in 4 M guanidine, 0.05 M Tris-HCl (pH 8.2), followed by chromatofocusing in a range from pH 8 to pH 5 in 6 M urea (Kluve-Beckerman et al., 1997). The final preparations are precipitated from ammonium sulfate (80% saturated), are dialyzed against water and lyophilized.

Induction of AA Amyloid Formation in Cell Culture

To induce the formation and deposition of amyloid AA in

macrophages / peritoneal cells in culture, recombinant mouse SAA2 (at a final concentration of 140 æg / ml) and AEF (at a final concentration of 12 æg / ml) were used. SAA 82 (7 μl) is added directly to the medium (350 μl) from a 7-10 mg / ml stock solution, prepared by dissolving purified recombinant SAA2 and lyophilized in 6 M urea, 25 mM HEPES, pH 7.2. The concentrations of stock solutions are adjusted after SDS-PAGE analysis and densitometric quantification of SAA bands stained with Coomassie blue. Stock solutions of AEF (2 mg / ml) are thoroughly mixed, to again suspend precipitated material before collecting an aliquot (2 μl) for addition to the culture medium (350 μl).

Cell Viability Assay

To confirm the cell viability of macrophage cultures following induction of AA amyloid (as described above) trypan blue exclusion is excluded in some cultures (days 5, 10 and 20). Prior to staining, the cells are rinsed 3 times with RPMI without serum. They are then covered for 1 minute with a solution of 2% (w / v) trypan blue in PBS (Perry et al., 1997).

Immediately after removal of trypan blue, the cells are fixed with 4% (w / v) paraformaldehyde, pH 7.5, for 10 minutes at room temperature, rinsed four times with PBS with gentle shaking, and examined under a microscope before staining with Congo red or Thioflavin S.

Phagocytic Activity of Macrophages

To evaluate and confirm the phagocytic activity of macrophages in culture (on days 5, 10 and 20 after induction

of AA amyloid), a latex bead capture assay is also employed. Blue stained polystyrene latex sticks, 0.8 Âμm in diameter (Sigma), are suspended in serum-free RPMI at a concentration of 7.2 x 83 108 / ml. Cells are rinsed 3 times with serum-free RPMI and then incubated for 30 minutes with 300 æl of the bead suspension. During incubation, the chamber slides are tightly wrapped in Parafilm and are gently shaken in a 37 ° C water bath. The bead suspension is removed and the cells are rinsed at least 10 times with RPMI. The cells are then fixed with formalin and stained with hematoxylin.

Congo Red Coloring

To confirm and quantify fibrillar AA amyloid deposition in culture, cells at 5, 10 and 20 days (post-induction of AA amyloid) are fixed in 100% ice-cold methanol and then stained for 45 minutes with Congo red, prepared in 80% alkaline ethanol (Putchler et al., 1962). After several rapid dips in water, the slides are immersed in hematoxylin for 2 minutes. The slides are then dipped once in 70% acidified ethanol, several times in water and once in a 1% NaOH solution. Dehydration is achieved by washing sequentially in 95% ethanol and 100% ethanol. The slides are cleaned in xylene and cover slides are applied using Permount. The extent of Congo red staining (i.e., red / green birefringence, visualized in polarized light) is determined quantitatively using an imaging system as described herein. Prior studies using this culture system indicated that the Congo red positive material remains bound to the chamber slides throughout the culture period (2-24 days) (Kluve-Beckerman et al., 1999). It has been previously demonstrated that accumulation of AA amyloid in the culture occurs within 2 days after the addition of SAA2 and AEF (Kluve-Beckerman et al., 1999). 84

Fluorescence with Thioflavin S

Fibrillary AA amyloid deposits are detected and analyzed after staining with Thioflavin S (Elghetany and Saleem, 1988). Thioflavin S fluorescence is quantitated using the image analysis system described herein.

Quantification of AA Amyloid% Load in Cell Culture by Image Analysis

To quantify the amyloid% loading present in cell cultures after formation and deposition of AA amyloid (at days 5, 10 and 20) we used a quantitative method involving image analysis. In summary, blades stained with Congo red or Thioflavin S are visualized in polarized light, or fluorescent light, respectively. 5-7 slides are used per treatment group, as described herein, to determine the% amyloid charge at a given magnification (X100). Quantitative analysis of images is performed using an Image Pro Plus (Media Cybernetics) imaging system connected to a Zeiss Axioskop 20 microscope (with polarization and fluorescence capabilities) through a CCD video camera. Images of fibrillar AA (polarized or fluorescent) amyloid sections are stored in a buffer, and a specific region (i.e., X100 magnification) is manually delineated and the total area of pixels occupied by the amyloid structures is determined. A monochromatic base limit is used to select pixels corresponding to polarized or immunofluorescent structures. The% of the region occupied by the tagged pixels (ie red / green to Congo red, yellow / green for Thioflavin S) is calculated. For all image analyzes, the state of treatment of cultures is unknown to the observer. Non-parametric Mann-Whitney analysis is performed using &quot; software &quot; Statview (SAS Institute, Cary, NC). Reductions of amyloid charges caused by treatment with polyhydroxylated aromatic compounds (as described below) can be determined quantitatively using the methodology described above. We successfully used this imaging system (using Congo red staining, fluorescence with Thioflavin S, or a biotinylated β-antibody) to determine amyloid burden, number of amyloid plaques, and plaque size in various regions of the brain in models of transgenic mice of Alzheimer's disease. Using this method we also quantified the number of macrophages in vitro (at an X100 magnification) that contain structures full of amyloid. Previous studies by Kluve-Beckerman et al. (1999) demonstrate that, using the cell culture system as described above, many of the macrophages accumulate fibrillar AA amyloid in the macrophage cytoplasm.

Test of Polyhydroxylated Aromatic Compounds Regarding Inhibition of Amyloid Formation and Deposition AA

Our data suggest that certain polyhydroxylated aromatic compounds possess potent amyloid-inhibiting activity. Disclosed below are polyphenolic compounds commercially available which are believed to be suitable for inhibiting the formation and deposition of fibrillar AA amyloid.

For structure-activity relationship studies, these readily available polyphenolic compounds with hydroxy groups located at various aromatic ring positions may be purchased from commercial sources. Initial studies to test the toxicity of these compounds indicate that they usually exhibit limited toxicity, even at high dosages. Quantification of the AA% amyloid filler as described herein is used to evaluate the ability of various polyhydroxy aromatic compounds to inhibit / destroy the formation and deposition of AA amyloid in culture.

Representative compounds disclosed and believed to be effective anti-amyloid and anti-alpha-synuclein / NAC inhibitors / destructors include EDTA (CioHi6N2C> 8 PM 292.2, used only as a negative control), and commercially available polyphenolic compounds with a variety of standards (C 15 H 14 O 6; MW 306.3), Epicatechin (C 15 H 14 O 6, MW 290.3), Catechin hydrate (C 15 H 14 O 6, MW 290.3), Epiglacatechin

Epigalhocatechin (C22H18O11; MW 458.4),

Epicatechin (C22H18O10; MW 442.4), Galhocatechin Gallate (C22H18O11; MW 458.4), Chlorogenic acid (C16Hi809; MW 354.3), Hematoxylin (C16H14O6; MW 302.3), Floroglucid (C12H10O5; MW 234.2) , Propyl gallate (C 10 H 12 O 5, MW 212.2), gallic acid ethyl ester (C 9 H 10 O 5, MW 198.2), gallic acid (C 7 H 6 O 5, MW 170.1), hydrate 3,4,5-trihydroxybenzamide (C6 H603, MW 126.1), ellagic acid (C14 H608, MW 302.2), Quercetin (C10 H12 O5 (C16 H60 NO2: MW 169.1), 5-hydroxydopamine (C8 H11 NO3, MW 205,6), 1,2,4-benzenetriol ; MW 338.3),

Pyrocatechol (C6H6 &lt;02; PM 110.1), Tannic acid (C76H52O46; MW 1701.2), Pyrogallol (C6H603; MW 126.1), and p-aminosalicylic acid (C7H7NO3; MW 153.1).

All of these compounds are commercially available from Sigma (St. Louis, MO), Fluka (St. Louis, MO) or Acros Organic (a subsidiary of Fisher Scientific, Palatine, IL). As examples of SAR-type studies, we compare compounds 87 containing three adjacent hydroxyl groups on an aromatic ring (examples include pyrogallol, 5-hydroxydopamine, gallic acid and propyl gallate) with compounds containing two adjacent hydroxyl groups (examples include pyrocatechol, quercetin and 1,2,4-benzenetriol). Compounds containing hydroxyl groups that are spaced at least 2 carbons (as florofluoride) are also evaluated in cell culture. These studies help to determine phenolic structural characteristics that contribute to the inhibition of AA fibrilogenesis formation and / or deposition. Also contemplated is the synthesis of chemical analogues of the most potent inhibitors identified herein. These studies provide good insights into the structural elements required for the observed bioactivity.

For cell culture studies a high initial dosage of test compounds (to be determined empirically) is used to determine if the compounds are able to inhibit the formation and / or deposition of amyloid AA, determined by methods as described below and here in another local. For these studies, each polyhydroxylated aromatic compound is added to cultures at the start of AA amyloid induction (i.e., administration of AEF + SAA 2). The cell culture media containing each individual test compound are replaced every 2-3 days. The% amyloid load is quantified on days 5 and 10 after AA amyloid induction and test compound treatment. Comparisons are made directly with% amyloid loading in cultures only induced for AA amyloid deposition (i.e., without test compound treatment). The identified compounds which appear to be promising as inhibitors of the formation and / or deposition of AA fibrillar amyloid at a single high dose are tested more extensively and at increasing concentrations (i.e., low dose, medium dose and high dose, to be determined empirically ) to confirm dose-dependent inhibition of formation and / or deposition of fibrillar AA amyloid. The selected compounds identified by screening using the cell culture methods described above are then tested in an experimental model of AA amyloidosis mouse. Figure 15 shows structure-activity relationship (SAR) studies of different polyhydroxylated aromatic compounds commercially available. Eight examples of the structures of different polyphenolic compounds available on the market, which differ in the location of the hydroxyl groups on the aromatic ring, are shown for use in SAR screening studies.

Quantification of AA Amyloid Using SDS-PAGE and Immunostaining In addition to the quantification of the% amyloid loading in culture after Congo red staining and fluorescence with Thioflavin E1. using imaging analysis, the accumulation of amyloid AA in the cell layer and media is determined using SDS-PAGE followed by immunostaining as previously described (Kluve-Beckerman et al., 1999). Briefly, the cell layers are rinsed three times with serum-free RPMI, are scraped to PBS, pelleted by centrifugation and solubilized in SDS sample buffer. For AA amyloid analysis in cell derived culture media, 15æl aliquots of media are added directly to SDS sample buffer. Triplicate samples are subjected to tricine-SDS-PAGE as previously described (Schagger and von Jago, 1987). Using this method, separate, spaced and stacked layers contain 16.5%, 10% and 4% polyacrylamide, respectively. A monoclonal antibody against the AA amyloid protein (Serotek) and the &quot; Western &quot; with enhanced chemiluminescence (ECL) according to the manufacturer's recommendations (Amersham Life Science). Using the methods described above, SAA2 usually appears as multiple bands at -12-14 kDa and higher, whereas AA protein appears as a -8.5 kDa main band (Kluve-Beckerman et al. 1999). Initially, once established cell cultures, N-terminal sequencing of the major band at -8.5 kDa is implemented to confirm the presence of the AA protein. We previously used the method of electrophoresis, electroelution and amino acid sequencing to identify other peptide sequences for other internal designs.

After establishing this system, we used scanning densitometry to determine the extent of amyloid AA protein in the cell layer and media after SAA2 + AEF induction (at 5 and 10 days after induction of amyloid AA), in the absence or presence of a high dose of each polyhydroxylated aromatic compound (as specified above). The mean of 3 scans per sample (with samples manipulated in triplicate SDS-PAGE) is used to determine AA amyloid protein levels in the cell layer and media under the various conditions. The statistical analysis comparing scanning densitometric analysis in the absence or presence of polyphenolic compounds is based on paired Student's t-tests with data presented as mean +/- 90 EP. Significance is reported at 95% (p <0.05), 99% (p <0.01) and 99.9% (p <0.001). ANOVA is also used, as needed.

Example 13

Identification of New Amyloid Inhibitors AA - Animal Studies

Studies suggest that certain polyhydroxylated aromatic compounds, such as epicatechin, appear to serve as potent inhibitors of the formation and deposition of AA fibrillar amyloid in vivo. We performed animal model studies to determine the efficacy of selected polyhydroxylated aromatic compounds in an experimental model of AA amyloidosis mouse. Groups of CBA / J mice (n = 8 per group) are first pretreated with different polyhydroxylated aromatic compounds, over a 7-day pre-treatment period, at a high non-toxic dose (to be determined empirically). The animals are induced to AA fibrillar amyloidosis by a single subcutaneous injection of AgN03 and a single injection in the tail vein of 100 μg AEF. Treatment during the amyloid induction phase with polyhydroxylated aromatic compounds continues for another 14 days prior to sacrifice (treatment time = 21 days in total). Spleens, livers and kidneys are obtained from each animal and the% amyloid load in each organ is quantified using image analysis. In addition, immunohistochemistry is implemented using antibodies against amyloid protein AA, SAA, heparan sulfate proteoglycans / glycosaminoglycans, amyloid P component and ApoE to evaluate the effects of test compounds on AA / SAA amyloid accumulation, and other cofactors known AA amyloids. 91

Methodology Treatment Groups

Groups (n = 8 per group) of 8 week old female CBA / J mice (Baxter Labs) are used for animal model studies. Initially, each animal is pretreated daily by oral gavage (in a volume of 0.5 ml) with a specific polyhydroxylated aromatic compound (selected from the test results above) at a high and non-toxic initial dose (to be determined empirically) over a 7-day pretreatment period. Typically, our low to high pre-dosages with orally administered polyhydroxyl aromatic compounds range from a low dose of 25 mg / kg / day to a high dose of 500 mg / kg / day. Equivalent dosages are used to compare different polyhydroxylated aromatic compounds taking into account the toxicity of the compound and the solubility of the compound (in PBS). Oral gavage is performed using a 22-gauge (Popper) oral dosing needle of test compounds dissolved in 0.50 mL of PBS (pH 7.4). At day 8, all animals are induced to AA amyloidosis by a single subcutaneous injection of 0.5 mL of 3% AgNCq (Fisher) in doubly distilled deionized water. A single injection in the tail vein of 100æg AEF in double distilled deionized distilled water follows. The animals are treated, for an additional 14 days, with each of the specific polyhydroxylated compounds. A group of 12 mice is induced for AA amyloidosis (by AgNO3 + AEF as described above) and is treated orally only with PBS (pH 7.4), in the absence of any test compounds. At the end of the 21 day treatment period, the animals are sacrificed by excessive dosing with 0.1 ml of eutosol, and the spleen, liver and kidneys of each animal are collected for staining and immunohistochemistry. The number of animals per group (n = 8 for test compounds, n = 12 for the saline control group) is determined from the data obtained and power calculations based on an alpha level of 0.05 (two sides) and deviations standard of 0.5, giving a power of 94%.

Production of AEF The AEF is prepared as described above.

Tissue Fixation and Preparation Tissue fixation and preparation are performed as previously described (Snow et al., 1991). In summary, the spleen, liver and kidneys are removed from each animal, fixed in 90% ethanol and 10% formaldehyde for 24 hours at -20 ° C, embedded in paraffin and sectioned at 25 μΜ (thick sections necessary for quantification of amyloid load, as described below). Cut approximately 50-75 sections per animal.

Congo Red staining and Thioflavin S Fluorescence The accumulation of AA fibrillar amyloid is detected by Congo red staining (Puchtler et al., 1962) and fluorescence with Thioflavin S (Elghetany and Saleem, 1988) as described above.

Quantification of Amyloid Load in Tissues

Image analysis is also used to quantify the% amyloid burden present in the spleen, liver and kidney of each animal. The loading of% amyloid in each organ is quantified as assessed after staining with Congo red (visualized in polarized light) or Thioflavin S (visualized in fluorescent light). 5-7 equally spaced slides per animal per spot are used treatment group) to determine the% amyloid load at a given magnification (X100). Quantitative analysis of the amyloid burden images in a given organ (and a specific amplification) is performed using the image analysis system described above. Potential reductions in amyloid load are determined quantitatively for each polyhydroxylated aromatic compound for each organ / animal. Certain test compounds are expected to reduce the amyloid burden by 40-60%.

Immunohistochemistry

Tissue sections are also immunostained to detect important markers of AA amyloid protein, and known amyloid associated AA cofactors. A monoclonal antibody against AA amyloid protein (Serotek) is used to conform to the location of AA amyloid deposits, while a polyclonal against SAA (Serotek) is employed to detect precursor protein, SAA. The presence of perlecan (a specific heparan sulfate proteoglycan implicated in AA amyloidosis) is detected (Snow et al., 1991) using a monoclonal antibody against &quot; core &quot; (Snow et al., 1994), while using a periplasmic fraction antibody generated by phage display (known as HS4C3, a generous offer of Dr. Van Kuppevelt) (van Kuppevelt et al., 1998) against a specific epitope of heparan sulfate GAG chains to determine the colocalization of heparan sulfate GAGs. The amyloid P component is detected using a polyclonal antibody (Research Diagnostics), while 94

ApoE is detected using a polyclonal antibody (Research Diagnostics).

For antibody localization we used standard fluorescence techniques (Basgen et al., 1989; Mosedale et al., 1996). The primary antibody for immunohistochemical staining is used over a series of dilutions to obtain the best specificity with the lowest background staining. Controls of positive immunostaining consist of sections treated with 1) pre-uptake of the antigen-over primary antibody (if available), 2) a different primary antibody of the same Ig class and species, and / or 3) PBS + 1% bovine serum albumin instead of the primary antibody (to ensure that there is no non-specific binding of the secondary antibodies employed).

Pharmacology and Utility

The disclosed compounds act to inhibit or prevent the formation of amyloid fibrils, inhibit or prevent the growth of amyloid fibrils, and / or cause disruption, destruction, and / or breakdown of amyloid fibrils and preformed amyloid protein deposits. Their activity can be measured in vitro by methods such as those discussed in Examples 1 to 4, while their in vivo activity against amyloidoses can be measured in animal models such as Alzheimer's disease, Parkinson's disease, systemic AA amyloidosis , type 2 diabetes and others.

The compounds also act to inhibit or prevent the formation of alpha-synuclein / NAC fibrils, inhibit or prevent the growth of alpha-synuclein / NAC fibrils, and / or cause disruption, destruction, and / or disintegration of fibrils of alpha-synuclein / NAC and preformed alpha-synuclein / NAC-associated protein deposits. Its activity can be measured in vitro by methods such as those discussed in Example 4 above. The therapeutic ratio of a compound can be determined, for example, by comparing the effective antifibrillar activity dose (antiamyloid or anti-alpha-synuclein / NAC in a suitable in vivo model in a suitable animal species, such as the mouse, to the dose giving rise significant weight loss (or other observable side effects) in the test animal species.

Pharmaceutical Compositions and Administration

In general, the compounds are administered in pure isolated form in therapeutically effective amounts in any of the usual manners known in the art, alone or in combination with at least one other compound and / or at least one other conventional therapeutic agent for the disease to be treated. A therapeutically effective amount may vary widely, depending on the disease, its severity, the age and relative health of the animal to be treated, the potency of the compound (s), and other factors. As antifibrillar agents, therapeutically effective amounts of the compounds may range from 10-1000 mg / kg body weight; for example, 10-100 mg / kg of body weight. The practitioner will achieve, without undue experimentation and considering his skill and this disclosure, to determine a therapeutically effective amount of a compound of this invention for the treatment of amyloidosis. 96

In general, the compounds are administered in the form of pharmaceutical compositions by one of the following routes: oral, topical, systemic (e.g., transdermal, intranasal, or suppository), or parenteral (e.g., intramuscular, subcutaneous, or intravenous injection) . The compositions may take the form of tablets, pills, capsules, semisolids, powders, sustained release formulations, solutions, suspensions, elixirs, aerosols, or any other suitable compositions; and comprise at least one compound in combination with at least one pharmaceutically acceptable excipient. Suitable excipients are well known to practitioners, and can be found, such as the methods of formulating the compositions, in common references, such as Alfonso AR: &quot; Remington's Pharmaceutical Sciences &quot;, 17th edition, Mack Publishing Company, Easton PA, 1985. Suitable liquid carriers, especially for injectable solutions, include water, aqueous saline, aqueous dextrose solution, and glycols.

In particular, the compound (s) may be orally administered, for example, in the form of tablets, troches, candies, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules , or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known in the art for the preparation of pharmaceutical compositions, and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives, to obtain pharmacologically elegant and palatable preparations. 97

Tablets contain the compound in admixture with non-toxic and pharmaceutically acceptable excipients which are suitable for the preparation of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example corn starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate or stearic acid or &quot; tale &quot;. The tablets may be uncoated or may be coated by known techniques to retard disintegration and absorption in the gastrointestinal tract and thereby provide prolonged action over a longer period of time. For example, a time delay material such as glycerol monostearate or glycerol distearate may be employed. Formulations for oral use may also be presented in the form of hardened gelatin capsules, wherein the compound is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or in the form of soft gelatin capsules, wherein the active ingredient is mixed with water or an oily medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the compound in admixture with suitable excipients for the preparation of aqueous suspensions. Such excipients consist of suspending agents, for example, carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum and acacia gum; dispersing or wetting agents may be naturally occurring phosphatides, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with aliphatic chain alcohols for example, heptadecaethylene oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids, such as hexitol, such as polyoxyethylene sorbitol mono-oleate, or condensation products of ethylene oxide with partial fatty acid esters and hexitol anhydrides, for example, polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, or one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the compound in a vegetable oil, for example, arachis oil, olive oil, sesame oil, or coconut oil, or in a mineral oil, as liquid paraffin. The oily suspensions may contain a thickening agent, for example, beeswax, paraffin or cetyl alcohol. Sweetening agents, such as those given below, and flavoring agents may be added to give a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant, such as ascorbic acid. Dispersible powders and granules suitable for the preparation of an aqueous suspension by addition of water provide the active ingredient in admixture with a dispersing or wetting agent, a suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already described above. Additional excipients, for example, flavoring and sweetening agents, may also be present.

The compounds may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil, for example, olive oil or arachis oils, or a mineral oil, for example, liquid paraffin, or mixtures thereof. Suitable emulsifying agents may consist of naturally occurring gums, for example, acacia gum or tragacanth gum, naturally occurring phosphatides, for example, soybean, lecithin, and occurring phosphatides, for example, soybean, lecithin, and esters or esters partial derivatives of fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion may also contain sweetening and flavoring agents. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents.

The compounds may also be administered by injection or infusion, subcutaneously or intravenously, or intramuscularly, intrasubnally, or intranasally, or by infusion techniques in the form of a sterile injectable or oleaginous suspension. The compound may be in the form of sterile injectable aqueous or oleaginous suspensions. These suspensions may be formulated according to the known area using suitable wetting dispersion agents and suspending agents which have been described above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a parenterally acceptable diluent or non-toxic solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Additionally, sterile fixed oils are conventionally employed as solvent or suspending medium. For this purpose, any soft fixed oils, including synthetic mono- or diglycerides, may be conventionally employed. In addition, fatty acids, such as oleic acid, have application in the preparation of injectables.

Dosage regimens may be adjusted to provide optimum therapeutic response. For example, several divided dosages may be administered daily, or the dosage may be proportionally reduced as indicated by the requirements of the therapeutic situation. It is especially advantageous to formulate the compounds in unit dosage forms for ease of administration and uniformity of dosage. Unit dosage forms, as used herein, refer to physically discrete units suitable as unitary dosages for the subjects to be treated; each containing a therapeutically effective amount of the compound and at least one pharmaceutical excipient. A pharmaceutical product will comprise a unit dosage form within a container which is labeled or accompanied by a label indicating the intended method of treatment, such as the treatment of an amyloid disease such as Alzheimer's disease, or a disease associated with fibril formation of alpha-synuclein, such as Parkinson's disease. A &quot; therapeutically effective dosage &quot; preferably inhibits amyloidosis or a disease associated with the formation of alpha-synuclein fibrils in a patient of at least 20, more preferably at least 40%, still more preferably at least 60%, and still more preferably at least 80% , relative to untreated subjects.

Preparation of Compounds

Many of the compounds used in disclosed methods are well known in the art and are commercially available. They may be briefly described in references such as &quot; Merck Index &quot;, 12th edition, Merck &amp; Co., Inc., Whitehouse Station, New Jersey, 1996 (which typically provides a reference for a synthesis or isolation), and can be found in chemical catalogs, such as those of commercial suppliers, as described herein.

INDUSTRIAL APPLICABILITY The invention has utility throughout the world for providing therapeutic relief and diagnostic assistance in the treatment of Parkinson's disease and Lewy body disease by the use of a catechin.

According to the statutes, the invention has been described in more or less specific language with respect to structural features. However, it should be understood that the invention is not limited to the specific features presented, since the presented means and construction comprise preferred forms of implementing the invention. Accordingly, the invention is claimed in any of its forms or modifications within the legitimate and valid scope of the appended claims, appropriately construed in accordance with the doctrine of equivalents.

Lisbon, July 27, 2012

Claims (9)

Use of a catechin in the preparation of a medicament for the treatment of Parkinson's disease or Lewy body disease, wherein the catechin is selected from the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins. 2. Catechin selected from the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins, for the treatment of Parkinson's disease or Lewy body disease. The use of claim 1, wherein the medicament is for oral administration, parenteral injection, intravenous injection, subcutaneous injection, intramuscular injection, topical administration, or administration by aerosol spray. The catechin of claim 2, which is intended for oral administration, parenteral injection, intravenous injection, subcutaneous injection, intramuscular injection, topical administration, or administration by aerosol spray. Synuclein method comprises amount of an alpha- or NAC fibrillogenesis treatment in an in vitro environment, wherein the method is the step of administering in the in vitro environment a therapeutically effective of a catechin 2 selected from the group consisting of catechin, epicatechin , galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins. A pharmaceutical composition for use in the treatment of Parkinson's disease or Lewy body disease comprising a therapeutically effective amount of a catechin selected from the group consisting of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and gallote of epicatechin, and pharmaceutically acceptable salts of the above catechins, and a pharmaceutically acceptable carrier, diluent or excipient, wherein the therapeutically effective amount of the catechin comprises a dosage in the range of about 0.1 to 500 mg / kg body weight of the subject. The composition of claim 6, wherein the therapeutically effective amount of the catechin comprises a dosage in the range of about 1.0 to 100 mg / kg body weight of the subject. sars The composition of claim 6 or claim 7, which comprises a mixture of two or more of the selected catechins which group consists of catechin, epicatechin, galhocatechin gallate, epigalhocatechin gallate, epigalhocatechin and epicatechin gallate, and pharmaceutically acceptable salts of the above catechins. 3 The composition of the claimed catechin claim is substantially pure. Lisbon, July 27, 2012 6, 7 or 8, in a way that isolated